Article
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Apoptosis, also known as programmed cell death, is a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation of multiples of 180-200 bp. Because apoptosis can be initiated not only by physiological stimuli but also by various chemical substances, the present paper investigates the suitability of apoptosis as a biomarker for biological effect monitoring in the marine environment. Aquarium experiments with dab (Limanda limanda) were carried out to examine the effects of exposure to cadmium, PCB 118, and PCB 77 (each 1 mg/kg fish wt) on apoptosis in dab liver. Determination of apoptosis was carried out by DNA gel electrophoresis and quantification of DNA fragments smaller than 1500 bp. In addition, accumulated amounts of cadmium, PCB 118, and PCB 77 in dab liver were analyzed. Quantification of the three xenobiotics resulted in an accumulation of about factor 10(2)-10(4). Exposure to PCB 118 and cadmium resulted in an increase in apoptotic DNA fragmentation. Exposure to PCB 77 led mainly to cell death by necrosis.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Metals: Aluminum Rainbow trout;Atlantic salmon; Common carp; Streaked prochilod Dussault et al., 2001;Monette and McCormick, 2008;Galindo et al., 2010;García-Medina et al., 2013;Razo-Estrada et al., 2013 Arsenic Grass carp; Japanese eel; Rainbow trout;Jarbua terapon;Hybrid tilapia; Zebrafish; Clearfin livebearer; Lake whitefish; Spotted snakehead; Indian walking catfish; Gilthead seabream Xiang et al., 2001;Celino et al., 2008;Raisuddin and Jha, 2004;Wang et al., 2004;Seok et al., 2007;Selvaraj et al., 2013;Pedlar et al., 2002;Roy and Bhattacharya, 2006;Datta et al., 2007;Guardiola et al., 2013;Cordero et al., 2014;Benhamed et al., 2016;Ghosh et al., 2006;Datta et al., 2009a, b;Nayak et al., 2007;Hermann and Kim, 2005 Pratap and Wendelaar Bonga, 1993;Iger et al., 1994;Brunelli et al., 2011;Lundebye et al., 1999;Berntssen et al., 2001;Piechotta et al., 1999;Rose et al., 2006;Okorie et al., 2014;Das et al., 2005;Chouchene et al., 2011;Migliarini et al., 2005;McClusky, 2006;Chan and Cheng, 2003;Hoole et al., 2003;Xiang and Shao, 2003;Risso-de Faverney et al., 2001;Gonzalez et al., 2006; Monteiro et al., 2009;Xiang et al., 2001;Mazon et al., 2002;Vergolyas et al., 2010;Luzio et al., 2013;Lundebye et al., 1999;Som et al., 2009;Hernandez et al., 2011;Feng et al., 2003;Nawaz et al., 2006;Krumschnabel et al., 2005 Piechotta et al., 1999;Zheng et al., 2016;Yadetie et al., 2014;Jianying et al., 2009;Wu et al., 2014;Cantrell et al., 1996;Cantrell et al., 1998;Hart et al., 1999;Dong et al., 2001;Dong et al., 2002;Toomey et al., 2001;Liu et al., 2007;Hagenaars et al., 2013;Deng et al., 2009;Birchmeier et al., 2005;Sweet et al., 1998;Zhang et al., 2008;Buckler et al., 2001 PAHs Mummichog; Channel catfish; Eel; Common carp; Pink snapper; Mulloway; Barramundi; Tilapia; Puffer fish Wang et al., 2010;Weber and Janz, 2001;Nigro et al., 2002;Reynaud et al., 2004;Reynaud and Deschaux, 2006;Bakhtyar and Gagnon, 2011;Holladay et al., 1998;Cao et al., 2013;Lu et al., 2009Lu et al., , 2010a Shi et al., 2011;Jin et al., 2011a;Cengiz, 2006;Calma et al., 2004;Eder et al., 2009;Piner and Üner, 2012;Connon et al., 2009 Neonicotinoids Olive flounder Su et al., 2007 Biopesticides Silver sea bream; Zebrafish Deane and Woo, 2005;Kari et al., 2007;Ishaq et al., 2017;Deane et al., 2006 observed at the apex of the intestinal folds of Atlantic salmon, Salmo salar (Lundebye et al., 1999). An increase of blast cell apoptosis in hematopoietic tissue during both lethal and sub-lethal Cu exposures was evidenced in Labeorohita (Som et al., 2009).The effects of copper exposure has been performed in both larval and adult zebrafish from toxicological point of view, and Hernandez et al.(2011) noted that the most sensitive organs to stress induced by waterborne copper were the central nervous system and the liver, even though the most affected in terms of cell death that is likely to be elicited by the induction of ROS, were the gills and head kidney. ...
... Metals: Aluminum Rainbow trout;Atlantic salmon; Common carp; Streaked prochilod Dussault et al., 2001;Monette and McCormick, 2008;Galindo et al., 2010;García-Medina et al., 2013;Razo-Estrada et al., 2013 Arsenic Grass carp; Japanese eel; Rainbow trout;Jarbua terapon;Hybrid tilapia; Zebrafish; Clearfin livebearer; Lake whitefish; Spotted snakehead; Indian walking catfish; Gilthead seabream Xiang et al., 2001;Celino et al., 2008;Raisuddin and Jha, 2004;Wang et al., 2004;Seok et al., 2007;Selvaraj et al., 2013;Pedlar et al., 2002;Roy and Bhattacharya, 2006;Datta et al., 2007;Guardiola et al., 2013;Cordero et al., 2014;Benhamed et al., 2016;Ghosh et al., 2006;Datta et al., 2009a, b;Nayak et al., 2007;Hermann and Kim, 2005 Pratap and Wendelaar Bonga, 1993;Iger et al., 1994;Brunelli et al., 2011;Lundebye et al., 1999;Berntssen et al., 2001;Piechotta et al., 1999;Rose et al., 2006;Okorie et al., 2014;Das et al., 2005;Chouchene et al., 2011;Migliarini et al., 2005;McClusky, 2006;Chan and Cheng, 2003;Hoole et al., 2003;Xiang and Shao, 2003;Risso-de Faverney et al., 2001;Gonzalez et al., 2006; Monteiro et al., 2009;Xiang et al., 2001;Mazon et al., 2002;Vergolyas et al., 2010;Luzio et al., 2013;Lundebye et al., 1999;Som et al., 2009;Hernandez et al., 2011;Feng et al., 2003;Nawaz et al., 2006;Krumschnabel et al., 2005 Piechotta et al., 1999;Zheng et al., 2016;Yadetie et al., 2014;Jianying et al., 2009;Wu et al., 2014;Cantrell et al., 1996;Cantrell et al., 1998;Hart et al., 1999;Dong et al., 2001;Dong et al., 2002;Toomey et al., 2001;Liu et al., 2007;Hagenaars et al., 2013;Deng et al., 2009;Birchmeier et al., 2005;Sweet et al., 1998;Zhang et al., 2008;Buckler et al., 2001 PAHs Mummichog; Channel catfish; Eel; Common carp; Pink snapper; Mulloway; Barramundi; Tilapia; Puffer fish Wang et al., 2010;Weber and Janz, 2001;Nigro et al., 2002;Reynaud et al., 2004;Reynaud and Deschaux, 2006;Bakhtyar and Gagnon, 2011;Holladay et al., 1998;Cao et al., 2013;Lu et al., 2009Lu et al., , 2010a Shi et al., 2011;Jin et al., 2011a;Cengiz, 2006;Calma et al., 2004;Eder et al., 2009;Piner and Üner, 2012;Connon et al., 2009 Neonicotinoids Olive flounder Su et al., 2007 Biopesticides Silver sea bream; Zebrafish Deane and Woo, 2005;Kari et al., 2007;Ishaq et al., 2017;Deane et al., 2006 observed at the apex of the intestinal folds of Atlantic salmon, Salmo salar (Lundebye et al., 1999). An increase of blast cell apoptosis in hematopoietic tissue during both lethal and sub-lethal Cu exposures was evidenced in Labeorohita (Som et al., 2009).The effects of copper exposure has been performed in both larval and adult zebrafish from toxicological point of view, and Hernandez et al.(2011) noted that the most sensitive organs to stress induced by waterborne copper were the central nervous system and the liver, even though the most affected in terms of cell death that is likely to be elicited by the induction of ROS, were the gills and head kidney. ...
... Despite the fact that intestinal epithelia provides a much durable barrier than the gill, the rates of apoptosis and cell proliferation in the intestine were more increased by dietary Cd (Lundebye et al., 1999;Berntssen et al., 2001). The exposure to Cd resulted in an increase in apoptotic DNA fragmentation and induced apoptotic cell death in the liver of dab (Limandalimanda;Piechotta et al., 1999) and in gut, gills and liver topsmelt(Atherinopsaffinis; Rose et al., 2006). Next to this tissues, also the muscles can evidence an accumulation, (in parrotfish, Oplegnathusfasciatus; Okorie et al., 2014). ...
... Metals: Aluminum Rainbow trout;Atlantic salmon; Common carp; Streaked prochilod Dussault et al., 2001;Monette and McCormick, 2008;Galindo et al., 2010;García-Medina et al., 2013;Razo-Estrada et al., 2013 Arsenic Grass carp; Japanese eel; Rainbow trout;Jarbua terapon;Hybrid tilapia; Zebrafish; Clearfin livebearer; Lake whitefish; Spotted snakehead; Indian walking catfish; Gilthead seabream Xiang et al., 2001;Celino et al., 2008;Raisuddin and Jha, 2004;Wang et al., 2004;Seok et al., 2007;Selvaraj et al., 2013;Pedlar et al., 2002;Roy and Bhattacharya, 2006;Datta et al., 2007;Guardiola et al., 2013;Cordero et al., 2014;Benhamed et al., 2016;Ghosh et al., 2006;Datta et al., 2009a, b;Nayak et al., 2007;Hermann and Kim, 2005 Pratap and Wendelaar Bonga, 1993;Iger et al., 1994;Brunelli et al., 2011;Lundebye et al., 1999;Berntssen et al., 2001;Piechotta et al., 1999;Rose et al., 2006;Okorie et al., 2014;Das et al., 2005;Chouchene et al., 2011;Migliarini et al., 2005;McClusky, 2006;Chan and Cheng, 2003;Hoole et al., 2003;Xiang and Shao, 2003;Risso-de Faverney et al., 2001;Gonzalez et al., 2006; Monteiro et al., 2009;Xiang et al., 2001;Mazon et al., 2002;Vergolyas et al., 2010;Luzio et al., 2013;Lundebye et al., 1999;Som et al., 2009;Hernandez et al., 2011;Feng et al., 2003;Nawaz et al., 2006;Krumschnabel et al., 2005 Piechotta et al., 1999;Zheng et al., 2016;Yadetie et al., 2014;Jianying et al., 2009;Wu et al., 2014;Cantrell et al., 1996;Cantrell et al., 1998;Hart et al., 1999;Dong et al., 2001;Dong et al., 2002;Toomey et al., 2001;Liu et al., 2007;Hagenaars et al., 2013;Deng et al., 2009;Birchmeier et al., 2005;Sweet et al., 1998;Zhang et al., 2008;Buckler et al., 2001 PAHs Mummichog; Channel catfish; Eel; Common carp; Pink snapper; Mulloway; Barramundi; Tilapia; Puffer fish Wang et al., 2010;Weber and Janz, 2001;Nigro et al., 2002;Reynaud et al., 2004;Reynaud and Deschaux, 2006;Bakhtyar and Gagnon, 2011;Holladay et al., 1998;Cao et al., 2013;Lu et al., 2009Lu et al., , 2010a Shi et al., 2011;Jin et al., 2011a;Cengiz, 2006;Calma et al., 2004;Eder et al., 2009;Piner and Üner, 2012;Connon et al., 2009 Neonicotinoids Olive flounder Su et al., 2007 Biopesticides Silver sea bream; Zebrafish Deane and Woo, 2005;Kari et al., 2007;Ishaq et al., 2017;Deane et al., 2006 observed at the apex of the intestinal folds of Atlantic salmon, Salmo salar (Lundebye et al., 1999). An increase of blast cell apoptosis in hematopoietic tissue during both lethal and sub-lethal Cu exposures was evidenced in Labeorohita (Som et al., 2009).The effects of copper exposure has been performed in both larval and adult zebrafish from toxicological point of view, and Hernandez et al.(2011) noted that the most sensitive organs to stress induced by waterborne copper were the central nervous system and the liver, even though the most affected in terms of cell death that is likely to be elicited by the induction of ROS, were the gills and head kidney. ...
... Metals: Aluminum Rainbow trout;Atlantic salmon; Common carp; Streaked prochilod Dussault et al., 2001;Monette and McCormick, 2008;Galindo et al., 2010;García-Medina et al., 2013;Razo-Estrada et al., 2013 Arsenic Grass carp; Japanese eel; Rainbow trout;Jarbua terapon;Hybrid tilapia; Zebrafish; Clearfin livebearer; Lake whitefish; Spotted snakehead; Indian walking catfish; Gilthead seabream Xiang et al., 2001;Celino et al., 2008;Raisuddin and Jha, 2004;Wang et al., 2004;Seok et al., 2007;Selvaraj et al., 2013;Pedlar et al., 2002;Roy and Bhattacharya, 2006;Datta et al., 2007;Guardiola et al., 2013;Cordero et al., 2014;Benhamed et al., 2016;Ghosh et al., 2006;Datta et al., 2009a, b;Nayak et al., 2007;Hermann and Kim, 2005 Pratap and Wendelaar Bonga, 1993;Iger et al., 1994;Brunelli et al., 2011;Lundebye et al., 1999;Berntssen et al., 2001;Piechotta et al., 1999;Rose et al., 2006;Okorie et al., 2014;Das et al., 2005;Chouchene et al., 2011;Migliarini et al., 2005;McClusky, 2006;Chan and Cheng, 2003;Hoole et al., 2003;Xiang and Shao, 2003;Risso-de Faverney et al., 2001;Gonzalez et al., 2006; Monteiro et al., 2009;Xiang et al., 2001;Mazon et al., 2002;Vergolyas et al., 2010;Luzio et al., 2013;Lundebye et al., 1999;Som et al., 2009;Hernandez et al., 2011;Feng et al., 2003;Nawaz et al., 2006;Krumschnabel et al., 2005 Piechotta et al., 1999;Zheng et al., 2016;Yadetie et al., 2014;Jianying et al., 2009;Wu et al., 2014;Cantrell et al., 1996;Cantrell et al., 1998;Hart et al., 1999;Dong et al., 2001;Dong et al., 2002;Toomey et al., 2001;Liu et al., 2007;Hagenaars et al., 2013;Deng et al., 2009;Birchmeier et al., 2005;Sweet et al., 1998;Zhang et al., 2008;Buckler et al., 2001 PAHs Mummichog; Channel catfish; Eel; Common carp; Pink snapper; Mulloway; Barramundi; Tilapia; Puffer fish Wang et al., 2010;Weber and Janz, 2001;Nigro et al., 2002;Reynaud et al., 2004;Reynaud and Deschaux, 2006;Bakhtyar and Gagnon, 2011;Holladay et al., 1998;Cao et al., 2013;Lu et al., 2009Lu et al., , 2010a Shi et al., 2011;Jin et al., 2011a;Cengiz, 2006;Calma et al., 2004;Eder et al., 2009;Piner and Üner, 2012;Connon et al., 2009 Neonicotinoids Olive flounder Su et al., 2007 Biopesticides Silver sea bream; Zebrafish Deane and Woo, 2005;Kari et al., 2007;Ishaq et al., 2017;Deane et al., 2006 observed at the apex of the intestinal folds of Atlantic salmon, Salmo salar (Lundebye et al., 1999). An increase of blast cell apoptosis in hematopoietic tissue during both lethal and sub-lethal Cu exposures was evidenced in Labeorohita (Som et al., 2009).The effects of copper exposure has been performed in both larval and adult zebrafish from toxicological point of view, and Hernandez et al.(2011) noted that the most sensitive organs to stress induced by waterborne copper were the central nervous system and the liver, even though the most affected in terms of cell death that is likely to be elicited by the induction of ROS, were the gills and head kidney. ...
... Despite the fact that intestinal epithelia provides a much durable barrier than the gill, the rates of apoptosis and cell proliferation in the intestine were more increased by dietary Cd (Lundebye et al., 1999;Berntssen et al., 2001). The exposure to Cd resulted in an increase in apoptotic DNA fragmentation and induced apoptotic cell death in the liver of dab (Limandalimanda;Piechotta et al., 1999) and in gut, gills and liver topsmelt(Atherinopsaffinis; Rose et al., 2006). Next to this tissues, also the muscles can evidence an accumulation, (in parrotfish, Oplegnathusfasciatus; Okorie et al., 2014). ...
... In mammal and invertebrate animal cell lines, for example, Cd has been shown to be responsible for disruption of the actin cytoskeleton after acute exposure over 24 h [13], and in molluskan hemocytes exposed to Cd in vitro, induction of MT mRNA is accompanied by the generation of reactive and potentially toxic oxygen species [14]. The influence of Cd on histopathological changes has been described in hepatic and pancreatic tissue of vertebrates: Studies in mouse [15] and fish liver [16,17] describe the occurrence of programmed cell death. Ultrastructural changes of the endoplasmic reticulum (ER) have been observed, for example, in rat liver [18,19] and mouse pancreatic acinar cells [20]. ...
... It has recently been shown that Cd disrupts cellular homeostasis in the mouse liver through its induction of programmed cell death and cell proliferation, with both being time-and dose-dependent [15,29]. Application of Cd (1 mg/kg fresh wt) has further been proposed to induce programmed cell death in fish liver (Limanda limanda) [16]. ...
Article
A sublethal dose of cadmium (Cd2+) administered via the diet during short-term exposure over 10 d induced programmed cell death in the hepatopancreas of the terrestrial pulmonate snail Helix pomatia. Condensed cell residues were predominantly phagocytosed by calcium cells, suggesting a specific function of these epithelial cells in metal detoxification or in clearing the organ of cellular debris from cell death. The considerable cell loss recorded by histological analysis was accompanied by enhanced cell proliferation. Intoxication with Cd was further associated with the pronounced abundance of residual bodies, predominantly recorded in excretory cells, and with pathological changes in the endoplasmic reticulum. During long-term Cd exposure, mortality increased with increasing Cd concentrations in the diet, as demonstrated by feeding experiments in the laboratory. Lethal effects of Cd appeared to be correlated with Cd overloading of the Cd-specific metallothionein isoform (Cd-MT), isolated and characterized previously from the animal's hepatopancreas. Stoichiometric analysis shows that the capacity of Cd-MT to bind six molar equivalents of Cd corresponds to a tissue Cd concentration of approximately 4 micromol/g dry weight. At this tissue concentration, all high-affinity metal-binding sites of Cd-MT are occupied by Cd2+. Cadmium exposure beyond this level gives rise to progressive destabilization of Cd-MT cluster structure in vitro, resulting in increasing proportions of weakly bound, or even unbound, Cd2+ ions. Our results suggest that in vivo, the observed overburdening of Cd-MT with Cd2+ reduces the viability of affected animals.
... Investigations of apoptotic induction in vivo are rare, especially concerning altered apoptotic rates in response to anthropogenic stressors. (Piechotta et al., 1999). Currently, tumor presence and tumor-associated lesions are used as biomarkers of carcinogenic environmental compounds at high concentrations; however, tumor formation is a late end-point indicator of environmental contamination. ...
... Changes in the level of apoptosis upon exposure to specific pollutants have been observed in numerous aquatic organisms such as; Gulf Killifish (Fundulus grandis) when exposed to N-methyl-N V -nitro-N-nitrosoguanidine (MNNG)(Blas-Machado et al., 2000 ), rainbow trout (Oncorhynchus mykiss) degenerating olfactory neuron and epithelial tissue when exposed to copper (Julliard et al., 1996; Lyons-Alcantara et al., 1998), and carp (Cyprinus carpio) epithelial cells dosed with cadmium (Iger et al., 1994). Investigation of complex contaminant mixtures has also demonstrated elevated levels of apoptosis in both white sucker (Catostomus commersoni) ovarian follicles affected by bleach kraft pulp mill effluent (Janz et al., 1997 ), and dab (Limanda limanda) experimentally treated with either cadmium or a mixture of PCB congeners 77 and 118 (Piechotta et al., 1999). Overall, numerous studies have suggested that aquatic organisms may exhibit alterations in the molecular regulation of apoptotic genes upon long-term exposure to various xenobiotic compounds or mixtures. ...
Article
Biomarkers are necessary for monitoring environmentally induced alterations at the molecular level in order to assess the impact of xenobiotic compounds on organism health. Apoptosis is a highly regulated cellular process that controls programmed cell death and is involved in tumor formation. Apoptosis thus may provide the basis for developing biomarkers for use in the field of ecotoxicology to monitor non-lethal levels of xenobiotic induced cellular stress and toxicity. This study shows that a brown bullhead (Ameiurus nebulosus) fibroblast cell line (BB-2) responds to known apoptotic inducers (staurosporine, cycloheximide, and tumor necrosis factor alpha (TNF-alpha)), as characterized by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick end-labelling (TUNEL). Furthermore, we characterized the apoptotic process using a series of newly identified bullhead genetic markers. Exposure to protein kinase C inhibitors altered the transcription of TF-cell apoptosis-related protein (TFAR)-15 and p23 with no effect on p53, inhibitor of apoptosis protein (IAP), or PNAS-2. Inhibition of protein synthesis caused a consistent reduction in the transcription of p53 and PNAS-2. This study demonstrates that our novel transcriptional markers are sensitive biomarkers for the study of the effects of xenobiotic chemicals on apoptosis in the brown bullhead.
... Apoptotic cell death naturally occurs throughout early development in teleost fishes as tissues differentiate, and it is a critical component of normal development and homeostasis (Kannan and Jain, 2000;AnvariFar et al., 2016;Elmore, 2007;Meier et al., 2000). However, inappropriate levels or timing of apoptosis induced by xenobiotic exposure or environmental stressors can lead to adverse biological effects (Kannan and Jain, 2000;Elmore, 2007;Datta et al., 2007;Piechotta et al., 1999;Zagarese and Williamson, 2001). In the present study, apoptotic fluorescence differed significantly among treatment groups (F = 72.58, ...
Article
Ultraviolet (UV) radiation can significantly increase the toxicity of polycyclic aromatic hydrocarbons (PAHs) in crude oil to early life stage (ELS) fishes through photo-induced /photo-enhanced toxicity. However, little is known about the sub-lethal effects and mechanisms of photo-induced PAH toxicity in ELS fishes. The present study investigated apoptosis and global transcriptomic effects in larval red drum (Sciaenops ocellatus) (24–72 h post-fertilization) following co-exposure to oil (0.29–0.30 μg/L ∑PAH50) and UV. Apoptosis was quantified using the TUNEL assay, and transcriptomic effects were assessed using RNA sequencing analysis. Apoptotic fluorescence was greatest in the eyes and skin following 24 and 48 h co-exposure to oil and UV, indicating photo-induced toxicity. Consistent with these phenotypic responses, pathways associated with phototransduction, eye development, and dermatological disease were among the top predicted pathways impacted. The present study is the first to provide global transcriptomic analysis of UV and oil co-exposure in an ELS fish.
... In some studies apoptosis and cell proliferation markers have been proposed as cellular biomarkers of Cd or other contaminant exposure in aquatic organisms (Ortego et al., 1995;Piechotta et al., 1999;Sweet et al., 1999). In fact, Berntssen et al. (2001) found regulated cell death and proliferation in the intestine of salmon (S. salar) fed dietary cadmium. ...
Article
The impact of cadmium on metabolism and osmoregulation was assessed in gilthead sea bream (Sparus aurata). Seawater acclimated fish were injected intraperitoneally with a sublethal dose of cadmium (1.25 mg Cd/kg body wt). After 7 days, half of the injected fish were sampled. The remaining fish were transferred to hypersaline water and sampled 4 days later. Gill and kidney Na(+)/K(+)-ATPase activities, plasma levels of cortisol, several metabolites and osmolytes, as well as osmolality were measured. Hepatosomatic index and condition factor were calculated. The expression levels of Na(+)/K(+)-ATPase, heat shock proteins (HSP70, HSP90) and proliferating cell nuclear antigen was assessed by western blotting. Cadmium treatment adversely affected the Na(+)/K(+)-ATPase activity, although, there was no perturbation in ion homeostasis and the animals were not compromised following transfer to hypersaline water. Increased cell proliferation and Hsp90 expression likely contributed to the attenuation of the deleterious effects of cadmium exposure.
... Apoptosis should be considered as a normal process by which cell population dynamics are correctly maintained (Waalkes et al., 2000 ).The presence of a few TUNEL-positive nuclei among proliferative cells, their lacking at the tip of the intestinal folds, and in particular, the finding of both TUNEL-positive and PCNA immunostained nuclei, suggests that, as in mammals, the control of cellular population dynamics in the L. aurata intestinal epithelium is located at the base of the folds, regulating proliferating cell number instead of differentiated non proliferating cells. A more extensive proliferative compartment is 337 found in the intestinal mucosa from polluted L. aurata specimens, as found in the gut of Atlantic salmon, following toxic levels of dietary heavy metal exposure (Lundebye et al., 1999; Berntssen et al., 2001;, or in some other organs, more commonly used for heavy metal pollution assessment (Piechotta et al., 1997; Kilemade et al., 2002; Feng et al., 2003; Lyons et al., 2004; Rissode Faverney et al., 2004; Mauceri et al. 2005).The increase of proliferative cells in adverse conditions allows us to hypothesize that a larger number of potential stem cells can begin to work, out of necessity , as found in mammals (Potten and Booth, 1997). Our results demonstrated that, not only is there an increase in the number of the S phase cells (strongly PCNA immunostained nuclei) spreading along the intestinal fold till its mid portion, but also of the G2/G1 phase cells (less immunostained PCNA nuclei) suggesting more rapid cell cycles. ...
Article
Full-text available
In the present paper, the effect of natural environment non- lethal heavy metal concentration on cell renewal of Liza aurata intestinal epithelium, was studied by the TUNEL (ter- minal deoxynucleotidyltransferase-mediated dUTP nick end labelling) method and anti-PCNA (proliferating cell nuclear antigen) immunohistochemistry, in order to detect, respec- tively, apoptosis and cell proliferation. In addition, the pres- ence and distribution of the cell renewal regulator, sero- tonin, was immunohistochemically investigated. In order to reduce variability, only immature specimens were consid- ered. The results indicated that in the control specimens from non-polluted areas, the PCNA immunoreactive nuclei of the proximal intestinal epithelium were only located at the bottom of the intestinal folds, together with a few TUNEL-positive nuclei, and goblet mucous differentiated cells. In the specimens from polluted areas, the number of PCNA immunoreactive cells was greatly enhanced, and they extended along the mid portion of the intestinal folds; the number of TUNEL-positive nuclei was enhanced as well, but they were almost exclusively detected in the third apical portion of the intestinal folds. Serotonin immunoreactive nerve elements were more frequently detected in the intes- tinal wall of L. aurata specimens from polluted areas, and besides that, some serotonin immunoreactive endocrine cells were also present. Variations in distribution and fre- quency of TUNEL-positive nuclei, PCNA immunoreactive nuclei, and serotonin immunoreactivity put in evidence an alteration of cell renewal with an enhancement of cell pro- liferation, probably leading to morphological intestinal fold changes.
... Apoptosis, also known as programmed cell death, is a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation of multiples of 180 Á/200 basepairs. Apoptosis could be demonstrated by an increased number of small DNA fragments in liver of dab exposed to PCBs and cadmium (Piechotta et al., 1999) and in ovarian follicular cells from prespawning white sucker exposed to BKME (Janz et al., 1997), thus indicating its possibility to be used as a biomarker of effects Many toxic chemicals cause strand breaks in DNA, either directly or indirectly. Alkaline unwinding and COMET assays are able to estimate the increase in the level of breaks above background resulting from exposure to these chemicals (Shugart, 1990a). ...
Article
Full-text available
In this review, a wide array of bioaccumulation markers and biomarkers, used to demonstrate exposure to and effects of environmental contaminants, has been discussed in relation to their feasibility in environmental risk assessment (ERA). Fish bioaccumulation markers may be applied in order to elucidate the aquatic behavior of environmental contaminants, as bioconcentrators to identify certain substances with low water levels and to assess exposure of aquatic organisms. Since it is virtually impossible to predict the fate of xenobiotic substances with simple partitioning models, the complexity of bioaccumulation should be considered, including toxicokinetics, metabolism, biota-sediment accumulation factors (BSAFs), organ-specific bioaccumulation and bound residues. Since it remains hard to accurately predict bioaccumulation in fish, even with highly sophisticated models, analyses of tissue levels are required. The most promising fish bioaccumulation markers are body burdens of persistent organic pollutants, like PCBs and DDTs. Since PCDD and PCDF levels in fish tissues are very low as compared with the sediment levels, their value as bioaccumulation markers remains questionable. Easily biodegradable compounds, such as PAHs and chlorinated phenols, do not tend to accumulate in fish tissues in quantities that reflect the exposure. Semipermeable membrane devices (SPMDs) have been successfully used to mimic bioaccumulation of hydrophobic organic substances in aquatic organisms. In order to assess exposure to or effects of environmental pollutants on aquatic ecosystems, the following suite of fish biomarkers may be examined: biotransformation enzymes (phase I and II), oxidative stress parameters, biotransformation products, stress proteins, metallothioneins (MTs), MXR proteins, hematological parameters, immunological parameters, reproductive and endocrine parameters, genotoxic parameters, neuromuscular parameters, physiological, histological and morphological parameters. All fish biomarkers are evaluated for their potential use in ERA programs, based upon six criteria that have been proposed in the present paper. This evaluation demonstrates that phase I enzymes (e.g. hepatic EROD and CYP1A), biotransformation products (e.g. biliary PAH metabolites), reproductive parameters (e.g. plasma VTG) and genotoxic parameters (e.g. hepatic DNA adducts) are currently the most valuable fish biomarkers for ERA. The use of biomonitoring methods in the control strategies for chemical pollution has several advantages over chemical monitoring. Many of the biological measurements form the only way of integrating effects on a large number of individual and interactive processes in aquatic organisms. Moreover, biological and biochemical effects may link the bioavailability of the compounds of interest with their concentration at target organs and intrinsic toxicity. The limitations of biomonitoring, such as confounding factors that are not related to pollution, should be carefully considered when interpreting biomarker data. Based upon this overview there is little doubt that measurements of bioaccumulation and biomarker responses in fish from contaminated sites offer great promises for providing information that can contribute to environmental monitoring programs designed for various aspects of ERA.
... However, it has been discussed, inclusively in flatfish, that certain contaminant interactions can actually impair the process, with prejudice to tissue recovery (e.g. Costa et al. 2010) and potentially leading to necrosis (Piechotta et al. 1999). In fact, many necrotic foci of variable extension were found in the hepatic parenchyma of animals exposed to contaminated sediments. ...
Article
Full-text available
The transcription of contaminant response-related genes was investigated in juvenile Senegalese soles exposed to sediments from three distinct sites (a reference plus two contaminated) of a Portuguese estuary (the Sado, W Portugal) through simultaneous 28-day laboratory and in situ bioassays. Transcription of cytochrome P450 1A (CYP1A), metallothionein 1 (MT1), glutathione peroxidase (GPx), catalase (CAT), caspase 3 (CASP3) and 90 kDa heat-shock protein alpha (HSP90AA) was surveyed in the liver by real-time PCR. CASP3 transcription analysis was complemented by surveying apoptosis through the TUNEL reaction. After 14 days of exposure, relative transcription was either reduced or decreased in fish exposed to the contaminated sediments, revealing a disturbance stress phase during which animals failed to respond to insult. After 28 days of exposure all genes' transcription responded to contamination but laboratory and in situ assays depicted distinct patterns of regulation. Although sediments revealed a combination of organic and inorganic toxicants, transcription of the CYP1A gene was consistently correlated to organic contaminants. Metallothionein regulation was found correlated to metallic and organic xenobiotic contamination in the laboratory and in situ, respectively. The transcription of oxidative stress-related genes can be a good indicator of general stress but caution is mandatory when interpreting the results since regulation may be influenced by multiple factors. As for MT1, HSP90 up-regulation has potential to be a good indicator for total contamination, as well as the CASP3 gene, even though hepatocyte apoptosis depicted values inconsistent with sediment contamination, showing that programmed cell death did not directly depend on caspase transcription alone.
... Chemical and viral-induced apoptosis are also well known, with the latter resulting from infection of host cells and thought to be a means to prevent the spread of infection [5]. In ®sh, apoptosis has been reported in many tissues, including the gill epithelium [6,7], skin [8], brain [9,10], liver [11,12], and a variety of lymphoid sites including blood, spleen, head kidney and thymus [3,13±16]. It is clear that not all leucocyte types are equally susceptible to induction of apoptosis [17], and this is as true of ®sh leucocytes as for other vertebrates. ...
Article
The rainbow trout caspase 6 gene has been cloned and sequenced. The open reading frame consisted of 906bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.
... Several earlier studies have reported that endocrine disrupting chemicals have been shown to induce apoptosis in a wide variety of cells including, PC 12 cells, thymocytes, and jurkat cells (Aoki et al., 2004;Yao et al., 2006Yao et al., , 2007. Environmental xenobiotics, such as Cd and PCBs exert their toxicity via apoptosis in fish liver (Piechotta et al., 1999). Recently, Weber et al. (2004) reported increased TUNEL positive hepatocytes in the liver of medaka (Oryzias latipes) exposed to 10 ng L 21 EE 2 from hatch to maturity in both sexes. ...
Article
Full-text available
Chalcalburnus tarichi is an endemic cyprinid fish living in the Lake Van basin located in the Eastern Turkey. Fish (3+ ages) were exposed to 17α-ethynylestradiol (EE(2); 1, 10, 100 ng L(-1); nominal concentrations) and nonylphenol (NP; 10, 60, 200 μg L(-1) ; nominal concentrations) for 32 days under semistatic daily renewal conditions. The exposure period was followed by an evaluation of liver and gonadal apoptosis and gonad histopathology in males and females. Exposure to the highest concentrations of EE(2) (100 ng L(-1)) and NP (200 μg L(-1) ) caused significant increases in the extent of apoptosis in liver and gonads. Treatment with 100 ng L(-1) of EE(2) and 200 μg L(-1) NP increased the number of TUNEL positive hepatocytes significantly in both sexes compared to controls. The rates of apoptosis in testicular germ cells and ovarian follicular cells were significantly greater at the same concentrations. Exposure to EE(2) (100 ng L(-1)) and NP (60 and 200 μg L(-1)) caused thickening of interstitial connective tissue (fibrosis) in the seminiferous tubule wall and testis-ova formation in males. In females treated with 100 ng L(-1) EE(2) , increased percentage of atretic ooctytes and fibrotic areas in the ovarian somatic stromal tissue were found in the ovaries. Increase in atresia, without a statistical significance, and fibrotic stromal tissue were also noted in 60 and 200 μg L(-1) NP treatments. Results suggest that EE(2) - and NP-dependent hepatotoxicity and gonadotoxicity are causally related to the increase in apoptosis in C. tarichi.
... Thus, PCBs may be cytotoxic by contributing either to apoptotic and/or necrotic cell death. Necrosis is a common outcome following an inflammatory response, and exposure to PCB 77 may mainly lead to cell death by necrosis (Piechotta et al., 1999). Our data clearly support the hypothesis that PCB 77, and possibly all environmental contaminants that function as Ah ligands, can contribute to an endothelial cell inflammatory response mediated mainly by an imbalance of the cellular oxidant/antioxidant status. ...
Article
Full-text available
Certain environmental contaminants such as polyhalogenated aromatic hydrocarbons may be implicated in diseases of the vasculature by compromising normal functions of vascular endothelial cells. We have shown previously that 3,3',4,4'-tetrachlorobiphenyl (PCB 77), an aryl hydrocarbon (Ah) receptor agonist, can cause disruption of endothelial barrier function. This was supported by an increase in oxidative stress as measured by enhanced 2',7'-dichlorofluorescein (DCF) fluorescence and activation of the oxidative stress-sensitive transcription factor NF-kappaB. We have now tested the protective effects of antioxidants vitamin E (alpha-tocopherol) and pyrrolidine dithiocarbamate (PDTC) on endothelial cell activation induced by PCB 77. Only vitamin E completely blocked PCB 77-mediated endothelial barrier dysfunction. This protective effect by vitamin E was associated with a decrease in both oxidative stress, as measured by DCF fluorescence, as well as in NF-kappaB activation. Furthermore, vitamin E decreased PCB 77-mediated production of the inflammatory cytokine IL-6. Although pretreatment of endothelial cells with PDTC prevented the induction of NF-kappaB by PCB 77, this inhibition was not associated with a decrease in DCF levels or protection against endothelial barrier dysfunction. Pretreatment with alpha-naphthoflavone (alpha-NF), an Ah receptor partial antagonist and specific inhibitor of cytochrome P450 1A, partially protected against PCB 77-induced endothelial barrier dysfunction. This observation was paralleled by the fact that alpha-NF did not fully antagonize the PCB-induced increase in DCF in endothelial cells. Furthermore, PCB-mediated induction of NF-kappaB and production of IL-6 were only partially blocked by alpha-NF. Of all the tested compounds (vitamin E, PDTC and alpha-NF), vitamin E was most potent in blocking PCB 77-mediated endothelial cell activation. These data give an insight into the potential use of vitamin E and related antioxidants to limit PCB-mediated cell injury and into the use of alpha-NF to explore mechanisms underlying the injurious potential of Ah receptor agonists.
... In the present study, apoptosis was frequently observed during follicular atresia, immediately downstream from the dam. According to Piechotta et al. (1999), this cell death process may be a sensitive biomarker for monitoring impacted habitats. Temperature and photoperiod are the two major environmental regulators of reproductive seasonality in teleosts, and the extent to which each fish species depends on temperature and photoperiod varies considerably, and may reflect the varied habitats and reproductive strategies (Blázquez et al., 1998). ...
Article
The curimatã-pacu Prochilodus argenteus is an important characiform from the São Francisco River basin that performs long-distance migrations for spawning upstream during the rainy season, when the temperature and photoperiod are elevated. Despite the interruption of the migratory routes by the Três Marias Dam and accentuated decline in fishing, the curimatã-pacu still sustains the fisheries at the Três Marias region in recent decades. The objective of this study was to evaluate the reproductive activity of P. argenteus in two sections of the São Francisco River, downstream from the Três Marias Dam, during the rainy season. In the first 34 km of the river, immediately below the dam, most of the females were in gonadal resting. At 34–54 km downstream from the dam, following the confluence with a medium-sized tributary, the Abaeté River, there was a high frequency of males and females in reproductive activity. Follicular atresia was more frequent in the upper section of the river while postovulatory follicles occurred predominantly in the lower section. Fulton's condition factor and gonadosomatic index indicated that the females were in a better physiological and reproductive condition below the confluence with the Abaeté River. In contrast to the females, the males were less affected by damming, and testicular maturation was largely achieved in two river sections. Thus, although the section of the São Francisco River immediately below the Três Marias Dam was found to be unfavourable for the reproduction of the migratory fishes due principally to the hypolimnetic water from the reservoir, reproductive success of P. argenteus was achieved below the Abaeté River. In this section, the species encountered appropriate conditions for maturation and spawning, i.e. warm temperatures above 24°C, high water flow and dissolved oxygen, and low water transparency. These results indicate the importance of a non-regulated tributary to minimize the ecological impact of a dam on the downstream native fish communities. Copyright © 2005 John Wiley & Sons, Ltd.
... Several studies have confirmed the formation of DNA strand breaks in dab following exposure to environmental contaminants. In vivo, an increase in the extent of DNA fragmentation has been demonstrated for exposure to PCBs 118 and 77 [28]. In the North Sea, differences in DNA strand break levels have been observed along a pollution gradient and positive correlations were obtained with certain PCB congeners (PCBs 28, 52, 101, 105, 118, 153, 138, 155 and 180) [29]. ...
Article
An in situ study of the relationship between marine contamination and genotoxic effects was performed on female dab (Limanda limanda) collected from different sites in the eastern English Channel (France) known to be contaminated by polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs). DNA adducts in liver and DNA strand breaks in blood cells were determined respectively by the nuclease P1-enhanced post-labelling technique and an alkaline version of the comet assay. The extent of DNA base oxidation was also assessed for three of the six sampling sites in the study, using a comet assay in combination with a specific DNA repair enzyme, formamidopyrimidine glycosylase (Fpg).With Comet data, two groups of sites that seem in accordance with the pollution level have been distinguished. The extent of DNA strand breaks was higher in adult than juvenile female dab. From a technical point of view, comet assay sensitivity was affected by high intra-individual variability that accounted for nearly 70% of total variance (the site factor represented no more than 26%). The combined use of the comet assay and Fpg showed the presence of DNA oxidised bases in environmentally exposed dab.Although qualitative differences between the sampling sites were observed in DNA adduct profiles, no significant differences were found for total DNA adduct levels. DNA adducts did not appear to be associated with PAH exposure. Histopathological studies showed hepatic steatosis in most of the animals examined. Only one pre-cancerous lesion (an early stage of hyperplasia) was detected (associated frequency of 0.8%).
... Occurrence of apoptosis can be detected by measuring the specific DNA fragmentation in histological sections using terminal dideoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Gavrieli et al., 1992; Tilly and Perez, 1997). Various studies have demonstrated increased cellular apoptosis in fish ovarian follicular cells, skin, medial yolk vein, thymocytes and hepatocytes after exposure to a variety of toxicants (Janz et al., 1997; Marty et al., 1997; Cantrell et al., 1998; Piechotta et al., 1999; Janz et al., 2001; Weber and Janz, 2001). Heat shock proteins are highly conserved proteins that are synthesized by the cells of all organisms studied so far (Jaattela, 1999; Whitley et al., 1999), including fish (Iwama et al., 1998). ...
Article
Previous studies exposing fish to xenoestrogens have demonstrated vitellogenin (VTG) induction, delayed gametogenesis, altered sex ratio, and decreased reproductive performance, with a majority of those studies focusing on exposure to single chemicals. The objective of this study was to determine the effects of binary mixtures of a weak estrogen receptor agonist, nonylphenol (NP) and a potent estrogen receptor agonist, 17alpha-ethinylestradiol (EE) on sex ratios, gametogenesis, VTG induction, heat shock protein 70 (HSP70) expression and reproductive capacity in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to nominal concentrations of 10 or 100 microg/l NP (NP10 or NP100, respectively), 1 or 10 ng/l EE (EE1 or EE10, respectively), 1 ng/l EE+10 or 100 microg/l NP (EE1+NP10 or EE1+NP100, respectively), 10 ng/l EE+10 or 100 microg/l NP (EE10+NP10 or EE10+NP100, respectively) or solvent control (0.01% acetone, v/v) in a static-renewal system with replacement every 48 h. At 60 dph, fish from each treatment were euthanized for histological examination of gonads, and whole body VTG and HSP70 levels. Remaining fish were reared in clean water until adulthood (240 dph) for breeding studies. In all EE10 exposure groups (EE10, EE10+NP10 and EE10+NP100), increasing NP concentration acted antagonistically to the action of EE in terms of VTG induction at 60 dph. Similarly, non-additivity was observed with egg production, where EE1+NP100 exposure resulted in significantly more eggs produced per breeding trial than EE1 alone. Histological staging of oogenesis revealed suppressed gametogenesis in an additive fashion in females at 60 dph. There were no differences among treatment groups in whole body HSP70 expression in 60 dph fish or in gonadal HSP70 expression in adult fish. Although there was no statistical evidence of non-additivity, breeding trials in adults revealed significant reductions in egg viability, egg hatchability and/or F1 swim-up success, suggesting that developmental exposures to xenoestrogens may cause irreversible effects on egg quality and progeny even after periods of depuration. In conclusion, these results suggest that environmentally relevant mixtures of NP and EE can produce additive or non-additive effects that depend on the particular response being determined and the respective exposure concentrations of each chemical.
... Differences in the defensive mechanisms of spiders should be reflected in their ability to counteract the effects of stressors, and can determine whether a given species can inhabit a metalpolluted environment. Oxidative stress caused by heavy metals may alter processes of cell death (Piechotta et al., 1999; Sweet et al., 1999; Mizutani et al., 2002; Pulido and Parrish, 2003). Whether the pathway leads to apoptosis or necrosis may depend on the ROS concentration (Raffray and Cohen, 1997). ...
Article
In the funnel web spider Agelena labyrinthica (Agelenidae; A. l.), sheet web spider Linyphia triangularis (Linyphiidae; L. t.) and wolf spider Xerolycosa nemoralis (Lycosidae; X. n.) from two differently polluted meadow sites in southern Poland, we studied the relations between antioxidant parameters (glutathione, GSH; glutathione peroxidases, GPOX, GSTPx; catalase, CAT; stress proteins-Hsp70, metallothioneins Mts), the intensity of apoptosis and necrosis, and heavy metal burdens of the midgut gland. Cellular reactions against stress caused by pollutants seemed to be sex-dependent. The concentrations of Zn and Cu in the midgut glands of male A. l. and X. n. were more than double that of the females, from both study sites. In male spiders from the heavily polluted site, both negative correlations (activity of caspase-3-like proteins vs Cu, Zn concentration; number of depolarized mitochondria vs Cu concentration) and positive correlations (number of necrotic cells vs Cu concentrations; activity of CAT vs Zn ) were noted. The defense of males against high metal content and its prooxidative effects is based mainly on GSH and CAT. In females the antioxidative reactions are species-specific and depend mainly on high peroxidase activity and on stress protein level. The increase in the number of apoptotic cells in the midgut gland of female spiders from the heavily polluted site suggests the defensive role of this process in maintaining the proper functioning of this organ.
... Ž lated cell death apoptosis Wagner et al., 1998; . Piechotta et al., 1999;Sweet et al., 1999 andcell Ž . proliferation Ortego et al., 1995 . ...
Article
Full-text available
Atlantic salmon (Salmo salar L.) parr were fed for one month on fish meal based diets supplemented with Cd (0, 0.7, or 204mg Cdkg–1 DW) or Cu (0, 34, or 691mg Cukg–1 DW) to assess the effects of non-essential (Cd) and essential (Cu) dietary metals on lipid peroxidation and the oxidative defence system. Cadmium accumulated significantly in the liver, intestine, and kidney of 204mg Cdkg–1exposed fish compared to controls. Copper accumulated significantly in the intestine, kidney, and liver of fish exposed to 691mg Cukg–1, and in the intestine of 34mg Cukg–1 exposed fish. Tissue Cu accumulation significantly increased intestinal and hepatic lipid peroxidation (as seen from thiobarbituric acid reactive substances, TBARS, levels) and subsequently decreased intestinal -tocopherol levels and increased intestinal and hepatic selenium dependent glutathione peroxidase (SeGSH-Px) activity. Dietary Cd significantly reduced SeGSH-Px activity in the intestine and liver of 204mg Cdkg–1 exposed fish compared to controls. No significant increase in tissue TBARS or reduction of -tocopherol levels was observed in the intestine of fish exposed to dietary Cd, with exception of the highest exposure group (204mg Cdkg–1). Dietary Cu caused depletion of tissue Se and glutathione levels, however the reduced availability of GSH and Se did not seem to explain the differences in SeGSH-Px activity. Dietary Cu had a direct effect on lipid peroxidation at a relatively low concentration (34mg Cukg–1). Cadmium indirectly affected tissue lipid peroxidation by damaging the oxidative defence system at the highest dietary concentration (204mg Cdkg–1).
... In the last decade, significant advances have been made in the discovery of apoptosis and the genes that control it. Apoptosis , also known as programmed cell death, is a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation, which can be initiated not only by physiological stimuli but also various chemical substances (Piechotta et al. 1999). Now it is clear that some oncogene mutations disrupt apoptosis, leading to tumor initiation, progression, and metastasis (Lowe and Lin 2000). ...
Article
We have identified two most significant biomarker genes, CYP1A1 (69.81 up-regulation) and MT1K (14.66 up-regulation), showing highest overexpression at p-value <0.005. These were selected out of several hundred genes induced in vitro, using PCB exposed human liver (HepG2) cells. Over expression of the CYP1A1 (cytochrome P450) gene was specific to PCB-77 and MT1K (Metallothionein) to PCB-153. Affymetrix oligonucleotide microarrays (mRNA) were used to screen the entire genome of human liver cells in a time-dependent exposure and were further validated by quantitative real-time RT-PCR.
... In the last decade, significant advances have been made in the discovery of apoptosis and the genes that control it. Apoptosis , also known as programmed cell death, is a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation, which can be initiated not only by physiological stimuli but also various chemical substances (Piechotta et al. 1999). Now it is clear that some oncogene mutations disrupt apoptosis, leading to tumor initiation, progression, and metastasis (Lowe and Lin 2000). ...
Article
Full-text available
Polychlorinated biphenyls (PCBs) are a group of persistent and widely distributed environmental pollutants that have various deleterious effects, e.g., neurotoxic, endocrine disruption and reproductive abnormalities, including cancers. Chronic exposure to environmentally hazardous chemicals like PCBs is of great concern to human health. It has been reported earlier that apoptotic proteins change in rats under chronic PCB treatment. It is of importance to determine if chronically exposed human cells develop a different protein expression. In the present study, the authors chronically exposed metabolically competent human liver (HepG2) cells at 50 to 100 microM to examine the role of the well-known environmentally hazardous pollutant non-coplanar 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) to study cell death. After 12 weeks of exposure these cells showed significant changes in apoptotic death in subsequent trypan blue growth assay, fluorescence microscopy, DNA fragmentation, and immunoblotting studies. Interestingly, chronically exposed cells showed marked differences in apoptotic and/or death-related proteins (e.g., Bcl2, Bak, and the pro and active forms of caspase-9, which were up-regulated), in contrast to acutely exposed (i.e., 48-h PCB-153 exposed) cells, which maintained linear growth despite repeated exposures. Similarly, tumor suppressor protein p53, proto-oncogene c-myc, and cell cycle regulator protein p21 were also up-regulated compared to nonchronically exposed HepG2 Cells. The results indicated that PCB-153-induced chronic exposure significantly altered different apoptotic (e.g., Bcl2, Bak, caspase-3) and tumor suppressor (e.g., p21, p53, and c-myc) proteins in the cellular model. These results suggest that chronic exposure to PCB-153 can induce cell survival by altering several apoptotic and tumor suppressor proteins.
... PCB exposure is associated with a wide array of acute toxic effects on fish including liver damages, impairment of osmoregulation, reduction of immune functions, reproductive dysfunctions, impairment of sexual maturation, developmental disturbances , apoptosis ATPase inhibition, altered retinoid homeostasis and mortality (Merkens and Kinter, 1971; Koch et al., 1972; Hansen et al., 1974; Nebeker et al., 1974; Svoboda et al., 1994; Monosson et al., 1994; Rice and Schlenk, 1995; Bills son et al., 1998; Kim and Cooper, 1999; Piechotta et al., 1999). PCBs can act as endocrine-disrupting chemicals that alter the behaviour of vertebrates as reviewed by Zala and Penn (2004). ...
Thesis
Full-text available
Diese Dissertation ist ein Beitrag zum Forschungsfeld der Stressökologie, im Spe-ziellen der Verhaltensökotoxikologie. Das spontane lokomotorische Verhalten der Fischarten Danio rerio und Leucaspius delineatus wurde unter sublethaler Expo-sition mit dem Cyanobakterientoxin Microcystin-LR (MC-LR) und dem Xenobio-tikums 2.4.4`-Trichlorobiphenyl (PCB 28) quantifiziert. Die Schwimmgeschwin-digkeit und Anzahl der Wendungen wurden kontinuierlich mit einem automati-schen Video-Monitoringsystem unter Laborbedingungen aufgezeichnet. In Hin-blick auf zyklische Aspekte wurden die Verhaltensanalysen mit chronobiologi-schen Methoden kombiniert. Hiermit wurde gezeigt, dass MC-LR und PCB 28 zu signifikanten Effekten in Verhalten und Aktivitätsrhythmik beider Fischarten führten. Höhere Konzentrati-onen beider Untersuchungssubstanzen verursachten eine deutliche Aktivitätsredu-zierung bei Danio rerio und Leucaspius delineatus. Einige der festgestellten Do-sis-Wirkungsbeziehungen entsprechen der Hormesistheorie, z. B. war bei geringe-ren MC-LR Konzentrationen ein Aktivitätsanstieg und bei höheren ein Aktivitäts-abfall beider Fischarten zu verzeichnen. Die Exposition mit MC-LR und PCB 28 verringerte bei beiden Testfischarten die Synchronisation der Aktivität mit dem Zeitgeber Licht. Dies führte bei beiden Fischarten zu einer Phasenverschiebung. Bei Leucaspius delineatus war unter dem Einfluss von MC-LR eine Phasenumkehr zu verzeichnen, die Fische wechselten von Tag- zu Nachtaktivität. Die Cosinor Analyse zeigte Dosis abhängige Veränderungen der circadianen Rhythmen der Schwimmaktivität (z.B. MESOR, Akrophase) unter Einfluss von MC-LR und PCB 28 an. Die Power Spektral Analyse indizierte für beide Fischar-ten unter Einwirkung von MC-LR and PCB 28 eine reduzierte Dominanz des cir-cadianen Rhythmuspeaks. Da die registrierten Unterschiede in der Reaktion beider Fischarten auf MC-LR und PCB 28 eher gering waren, sind Ergebnisse der Art Danio rerio, die häufig in Toxizitätstests verwendet wird, mit denen der einheimischen Art Leucaspius deli-neatus vergleichbar. Die Ergebnisse belegen, dass Verhaltensuntersuchungen in Kombination mit chronobiologischen Auswertemethoden eine sensitive und zuverlässige Abschät-zung des Gefährdungspotentials von Substanzen sowohl auf dem Gebiet der Öko-toxikologie als auch für Biomonitoring ermöglichen.
... Stress-induced changes in apoptosis were investigated specially in regard to the immune-neuroendocrine axis (Alford et al. 1994;Harris and Bird 2000;Yamashita et al. 2003). In addition, the suitability of apoptosis as a biomarker for biological effect monitoring in the marine environment has been discussed (Piechotta et al. 1999;Solé et al. 2013). In general, salinity, temperature, pH, oxygen level and UV radiation are regarded as the main environmental stressors for fish. ...
Article
Full-text available
Apoptosis, a form of programmed cell death, is a critical component in maintaining homeostasis and growth in all tissues and plays a significant role in immunity and cytotoxicity. In contrast to necrosis or traumatic cell death, apoptosis is a well-controlled and vital process characterized mainly by cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane blebbing and apoptotic bodies. Our understanding of apoptosis is partly based on observations in invertebrates but mainly in mammals. Despite the great advantages of fish models in studying vertebrate development and diseases and the tremendous interest observed in recent years, reports on apoptosis in fish are still limited. Although apoptotic machinery is well conserved between aquatic and terrestrial organisms throughout the history of evolution, some differences exist in key components of apoptotic pathways. Core parts of apoptotic machinery in fish are virtually expressed as equivalent to the mammalian models. Some differences are, however, evident, such as the extrinsic and intrinsic pathways of apoptosis including lack of a C-terminal region in the Fas-associated protein with a death domain in fish. Aquatic species inhabit a complex and highly fluctuating environment, making these species good examples to reveal features of apoptosis that may not be easily investigated in mammals. Therefore, in order to gain a wider view on programmed cell death in fish, interactions between the main environmental factors, chemicals and apoptosis are discussed in this review. It is indicated that apoptosis can be induced in fish by exposure to environmental stressors during different stages of the fish life cycle.
... Apoptosis, also known as programmed cell death, is a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation of multiples of 180 Á/200 basepairs. Apoptosis could be demonstrated by an increased number of small DNA fragments in liver of dab exposed to PCBs and cadmium (Piechotta et al., 1999) and in ovarian follicular cells from prespawning white sucker exposed to BKME (Janz et al., 1997), thus indicating its possibility to be used as a biomarker of effects Many toxic chemicals cause strand breaks in DNA, either directly or indirectly. Alkaline unwinding and COMET assays are able to estimate the increase in the level of breaks above background resulting from exposure to these chemicals (Shugart, 1990a). ...
... Apoptosis, also known as programmed cell death, is a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation of multiples of 180 Á/200 basepairs. Apoptosis could be demonstrated by an increased number of small DNA fragments in liver of dab exposed to PCBs and cadmium (Piechotta et al., 1999) and in ovarian follicular cells from prespawning white sucker exposed to BKME (Janz et al., 1997), thus indicating its possibility to be used as a biomarker of effects Many toxic chemicals cause strand breaks in DNA, either directly or indirectly. Alkaline unwinding and COMET assays are able to estimate the increase in the level of breaks above background resulting from exposure to these chemicals (Shugart, 1990a). ...
... An increase in apoptosis, an energetically costly process of programmed cell death, frequently induced by ROS signaling (Robertson and Orrenius, 2000), has also been observed. For example, Cd induced apoptotic DNA fragmentation or apoptotic cell death in dub (Limanda limanda) (Piechotta et al., 1999), Atlantic salmon (Berntssen et al., 2001) and topsmelt (Rose et al., 2006). ...
Article
Full-text available
Adverse outcome pathways (AOPs) organize knowledge on the progression of toxicity through levels of biological organization. By determining the linkages between toxicity events at different levels, AOPs lay the foundation for mechanism-based alternative testing approaches to hazard assessment. Here, we focus on growth impairment in fish to illustrate the initial stages in the process of AOP development for chronic toxicity outcomes. Growth is an apical endpoint commonly assessed in chronic toxicity tests for which a replacement is desirable. Based on several criteria, we identified reduction in food intake to be a suitable key event for initiation of middle-out AOP development. To start exploring the upstream and downstream links of this key event, we developed three AOP case studies, for pyrethroids, selective serotonin reuptake inhibitors (SSRIs) and cadmium. Our analysis showed that the effect of pyrethroids and SSRIs on food intake is strongly linked to growth impairment, while cadmium causes a reduction in growth due to increased metabolic demands rather than changes in food intake. Locomotion impairment by pyrethroids is strongly linked to their effects on food intake and growth, while for SSRIs their direct influence on appetite may play a more important role. We further discuss which alternative tests could be used to inform on the predictive key events identified in the case studies. In conclusion, our work demonstrates how the AOP concept can be used in practice to assess critically the knowledge available for specific chronic toxicity cases and to identify existing knowledge gaps and potential alternative tests.
... The increase of ROS level also contribute to DNA damage, which results in disorder of normal cell function, leading to death [24]. Research on apoptosis, considered now as an indicator of chronic oxidative stress [25], are concentrated around the mitochondria, which are the main source of ROS. Currently it is assumed that ROS by oxidative stress mediates apoptosis [26]. ...
Article
In the present study, the investigation of the effect of chlorfenvinphos (CFVF) on necrotic and apoptotic changes as well as on selected morphological and biochemical parameters in human blood mononuclear cells were investigated.We analyzed the effect of this compound on proteins damage and free-radical formation in human blood mononuclear cells. Furthermore, changes in the size (FSC-A) and granularity (SSC-A) of human blood mononuclear cells exposed to chlorfenvinphos were assessed. In order to detect apoptosis, two testes were used including analysis of YO-PRO-1 iodide/propidium iodide and Annexin V/propidium iodide staining, which revealed that chlorfenvinphos increased the number of apoptotic cells at its highest concentration of 250 μM. Chlorfenvinphos at the concentrations from 50 and 100 μM increased the size and granularity of the blood mononuclear cells, respectively. Moreover, chlorfenvinphos induced the statistically significant loss of human blood mononuclear cells viability at the concentration of 250 μM. Protein damage (the increase in carbonyl groups content) was provoked by CFVF at concentrations of 100 μM and 250 μM. Furthermore, chlorfenvinphos from relatively low concentrations of 5 μM induced reactive oxygen species formation (ROS).Conclusion: The present findings provide information that chlorfenvinphos only at 250 μM is harmful to human blood mononuclear cells, the concentration which may appear in the organism only as a result of acute or subacute poisoning. Lower concentration (5–50 μM), which caused changes in level of ROS formation can affect human organism as a result of environmental exposure.
... Both, in vitro and in vivo studies have shown that gonadotropins, 17-ß oestradiol and the epidermal growth factor act as apoptosis suppressers in preovulatory follicles of the rainbow trout Oncorhynchus mykiss (Walbaum) Wood & Van Der Kraak, 2002). Recent studies indicate apoptosis as a biomarker of environmental impact, since fishes exposed to xenobiotics have shown a decrease in the gonado-somatic index associated with an increase in the apoptosis rate Piechotta et al., 1999;Weber et al., 2002). ...
Article
Involution and resorption of both postovulatory and atretic follicles were analysed in piau‐jejo Leporinus taeniatus(Characiformes, Anostomidae) in order to evaluate the role of apoptosis during ovarian regression. Histological and ultrastructural analyses showed hallmarks of apoptosis in the granulosa: aggregation of compacted chromatin against the nuclear envelope, cell shrinkage, surface blebbing, loss of cell adhesion and cell fragmentation into apoptotic bodies. Protein synthesis activity preceded the onset of the cell death. The breakdown of the basement membrane led to the detachment of the granulosa cells into the follicular lumen. TUNEL‐positive reactions were detected in in situ DNA fragmentation of granulosa of both postovulatory and atretic follicles. Apoptosis increased in a time‐dependent manner contributing to reduction of the follicular areas. The apoptotic index (per cent of apoptotic cells) of the granulosa increased in postovulatory follicles soon after spawning, then these follicles degenerated and only remnants were observed at 7 days. In contrast, the granulosa cells reabsorbed the yolk during follicular atresia and the apoptotic index increased only in the late stage of regression. The results indicated apoptosis as the major mechanism to rapidly eliminate postovulatory follicles and being an essential process in the ovarian regression after spawning.
Article
In this study, we used an integrated approach to determine whether key biochemical, cellular, and physiological responses were related to growth impairment of cadmium (Cd)-exposed larval topsmelt (Atherinops affinis). Food intake (Artemia franciscana nauplii), oxygen consumption rates, apoptotic DNA fragmentation (TUNEL assay), and metallothionein (MT)-like protein levels, were separately measured in relation to growth of larval topsmelt aqueously exposed to sublethal doses of Cd for 14 days. Cadmium accumulation and concentrations of abundant metals were also evaluated in a subset of fish. Fish in the highest Cd treatments (50 and 100 ppb Cd) were smaller in final mean weight and length, and consumed fewer A. franciscana nauplii than control fish. Food intake was positively correlated with final weight of larval topsmelt in Cd and control treatments; food intake increased as final weight of the fish increased. Oxygen consumption rates were positively correlated with Cd concentration and mean oxygen consumption rates were inversely correlated with final mean weight of topsmelt; the smallest fish were found in the highest Cd treatment and were respiring at higher rates than control fish. Apoptotic DNA fragmentation was concentration-dependent and was associated with diminished growth. Apoptotic DNA fragmentation was elevated in the gill of fish exposed to 50 ppb Cd, and in the gut, gill, and liver of fish exposed to 100 ppb Cd. Metallothionein (MT)-like protein levels in fish from 100 ppb Cd treatments were significantly higher than those in other treatments. Oxygen consumption rates may have increased as a compensatory response to Cd exposure. However, it is likely that the energy produced was allocated to an increased metabolic demand due to apoptosis, MT synthesis, and changes in ion regulation. This diversion of energy expenditures could contribute to growth impairment of Cd-exposed fish.
Article
RAMP embraces the integrated use of methods for the rapid measurement, assessment and access to information on the nature, sources and influences of coastal environmental change. It embraces approaches held in the literature, research and programs of RAMP (Rapid Assessment of Marine Pollution) and the emerging work described as RASE (Rapid Assessment of Socio-Economic Indicators). To protect coastal ecosystems and the health of communities effectively, management infrastructure requires the tools and resources necessary to detect damage to coastal ecosystems and their components, identify causative agents, impose remedial action, and demonstrate that measures have been effective. Pragmatic monitoring and prediction capabilities must also be built to provide further confidence that human impacts are being minimized and that threats to human health have been contained. For most of the world, however, the ability to build such capability is a technical challenge and often cost prohibitive. These constraints point to the need to develop and expand the integrated use of simple, robust, cost-effective environmental assessment procedures. This paper suggests that a system built around the Rapid Assessment of Marine Pollution (RAMP) and the Rapid Assessment of Socio-Economic Indicators (RASE) can, should and in some cases already has been effective in meeting such informational and management needs.
Article
PCBs are persistent environmental contaminants that cause a variety of adverse health effects in wildlife and humans. This article describes the use of signature gene expression patterns that link increased PCB exposure with progressive, adverse biological effects. Developing Xenopus laevis tadpoles of two age classes were exposed to the PCB mixture Aroclor 1254 for 2 days. Real-time PCR was used to quantitate mRNA expression for 11 physiologically relevant, potential bioindicator genes. Younger tadpoles (5 days postfertilization) were resistant to Aroclor 1254 and showed few changes in gross morphology, swimming behavior, survival, or gene expression. Older tadpoles (11 days postfertilization) were more susceptible to Aroclor 1254. Exposure to 25 and 50 ppm Aroclor 1254 caused alterations in gross morphology and swimming behavior and statistically significant decreases in survival. These tadpoles showed statistically significant decreases in gene expression for 9 out of the 11 genes measured. Tadpoles exposed to 10 ppm showed incipient health changes but had gene expression profiles similar to the tadpoles treated with higher doses of Aroclor 1254. Tadpoles exposed to 1 ppm did not exhibit any observable adverse health effects, yet statistically significant decreases in gene expression occurred in these tadpoles (4 out of 11 genes). After prolonged exposure, tadpoles exposed to 1 and 10 ppm Aroclor 1254 exhibited health effects similar to those exposed to higher concentrations. Therefore, changes in expression of specific genes may serve not only as molecular bioindicators of Aroclor 1254 exposure but also as predictors of impending adverse health effects.
Article
Pollutants found dispersed in water can cause irritations on the gills, challenge the immune system and prejudice the welfare of the fish. Here we investigated molecules linked to proliferation, survival, and cell death, as well as inflammatory and vascular control, in a model of fish gill remodeling, from injury to recovery. We assessed the gill histology and immunohistochemistry for PCNA, iNOS, HSP70, and Bax in Hypostomus francisci obtained from a river subjected to chronic anthropic influences and then after they were placed in water of good quality. A total of 30 H. francisci adult individuals were collected and distributed into two groups: euthanized on the day of capture (group 1) and maintained for 30 days in an aquarium (group 2). In all the fish from group 1, the primary and secondary lamellae showed hypertrophy of the respiratory epithelium, lamellar fusion, lifting of the epithelium, aneurysm, hyperemia, and vascular congestion. On the other hand, in all the fish from group 2, restoration of gill integrity was observed, and the primary and secondary lamellae showed a simple epithelium, absence of lamellar fusion, hypertrophy, and aneurysm. Gills of fish from group 1 had higher frequency of cells immunopositive for PCNA, iNOS, HSP70, and Bax than those of fish from group 2 (p < 0.05). The molecular and cellular mechanisms from injury to recovery were proposed, with a balance between survival and cell death signals being essential for determining the gill structure. In addition, the findings indicate that recovery of the structural organization of gills is possible if fishes are maintained in good-quality water, indicating the importance of the conservation of aquatic environments.
Article
A novel caspase recruitment domain protein (CARD) was isolated from common carp Cyprinus carpio L. by expressed sequence tag analysis. This gene consist of a 2016 bp open reading frame and untranslated regions, which is putatively translated to a protein of 535 amino acid residues. The gene harbors domains (CARD and Coiled-coil domain), which are conserved in proteins of CARD family. The CARD domain have carp was similar to human CARD9 with 72.4% identity. Expression analysis revealed that CARD gene of carp (carp-CARD) expressed in normal tissues of head kidney, spleen, liver, heart and brain. Here we demonstrated that the expression of carp-CARD increased by cortisol treatment in all the tissues and had a high and long lasting expression in cortisol treated spleen.
Article
The aim of this study was to evaluate the relations between apoptosis and the activity of antioxidant enzymes (superoxide dismutase; catalase) and quantitative changes in stress protein positive cells (Hsp70; metallothionein) in midgut glands of funnel web spiders Agelena labyrinthica (Agelenidae) and wolf spiders Pardosa lugubris (Lycosidae) exposed to high temperature and pesticide under laboratory conditions. The spiders were collected from two meadow ecosystems differently polluted with metals (Olkusz and Pilica, southern Poland). Under stress conditions, P. lugubris had fewer apoptotic cells in the midgut glands than A. labyrinthica. In P. lugubris from both sites, the observed increase in the percentage of metallothionein and Hsp70-positive cells, simultaneous with intensification of superoxide dismutase and catalase activity, suggests an anti-apoptotic function of those proteins in representatives of wandering spiders. In the midgut glands of A. labyrinthica, heat shock and dimethoate increased the number of Annexin V-positive cells as well as the amounts of mitochondria with low transmembrane potential (DeltaPsi(m)) versus the control. The changes in the percentage of MT- and Hsp70-positive cells in funnel web spiders were less than in wolf spiders. The absence of change in SOD and CAT activity in A. labyrinthica shows that the participation of those enzymes in antioxidant reactions is minimal in this species.
Article
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyl (PCB) 126 produce thymic atrophy and immunosuppression. This study explored the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 is associated with an increase in apoptotic thymocytes at the end of incubation in chicken embryos. Eggs were injected via the air cell with PCB 126 (0.05, 0.13, 0.32, 0.64, and 0.80 ng/g egg) on d 0 of incubation, and tissues were collected on d 20. Controls included noninjected and vehicle-injected (sunflower oil) eggs. Thymocytes were cultured for 6 h and analyzed by flow cytometry for decreased DNA content (propidium iodide staining) and cell size (forward scatter), which indicate apoptosis. PCB 126 induced dose-dependent mortality with an LD50 of 1.01 ng/g and lowest-observed-effect concentration (LOEC) of 0.32 ng/g. Teratogenic effects commonly associated with TCDD and planar PCBs, including cranial and foot deformities and subcutaneous edema, tended to increase with dose of PCB 126. PCB 126 reduced thymus mass by approximately 20% at 0.64 and 0.8 ng/g, the number of viable thymocytes by approximately 20-24% at and above 0.13 ng/g, and the number of bursal lymphoid cells by 57% at 0.64 ng/g. The percentage of apoptotic thymocytes increased with dose, reaching levels 2 times greater than controls at 0.8 ng/g. Electrophoresis of low-molecular-weight DNA from thymocytes of all doses demonstrated fragments in multiples of 180 bp. This DNA laddering is a hallmark of apoptosis. At all doses, thymocytes exhibited caspase-3 activation, another indicator of apoptosis. The results of this experiment supported the hypothesis that the thymic atrophy produced by developmental exposure to PCB 126 in chicken embryos is associated with an increase in apoptotic thymocytes on embryonic d 20.
Article
In marine molluscs, the epithelium of the digestive gland is composed of two cell types, namely, digestive and basophilic cells. Under normal physiological conditions digestive cells outnumber basophilic cells, but under different stress situations the composition of the epithelium changes, basophilic cells apparently replace digestive cell. Winkles, Littorina littorea, were exposed to 1.25mg/l Cd for 20 days to provoke cell type replacement. Then, animals were depurated in clean seawater for 10 days to determine whether cell type replacement was reversible. Digestive glands were fixed in Carnoy and paraffin embedded for histological analysis. The volume densities of basophilic cells (Vv(BAS)) and digestive cells (Vv(DIG)) were calculated by stereology on hematoxylin-eosin stained sections. Vv(BAS) increased and Vv(DIG) decreased in Cd-exposed animals. After estimation of cell size and absolute cell numbers, these changes were attributed to digestive cell loss and concomitant basophilic cell hypertrophy but not to increased numbers of basophilic cells. Cell type composition and cell size almost fully returned to normal values after 10-day depuration. Accordingly, PCNA immunohistochemistry demonstrated that proliferating digestive cells were more abundant in winkles exposed to Cd and after 10-day depuration than in control specimens, suggesting that net digestive cell loss was accompanied by increased digestive cell proliferation. Thus, Cd-exposure seems to provoke an enhanced digestive cell turnover in order to cope with Cd detoxification. Intralysosomal accumulation of metals (autometallographied black silver deposits; BSD) was used as a biomarker of exposure to Cd and lysosomal structural changes as an effect biomarker to see whether cell type composition might have any effect on these endpoints. BSD formed around Cd ions, in digestive cell lysosomes of Cd-exposed winkles whereas basophilic cells appeared devoid of them. After depuration, BSD were less conspicuous. Enlarged lysosomes were observed in Cd-exposed winkles, lysosome size returning to control levels after 10-day depuration. Changes in digestive cell proliferation, digestive cell loss and basophilic cell hypertrophy did not apparently affect the biomarkers investigated herein.
Article
Full-text available
An immense amount of data is available on biomarkers related to different eco-toxicants. But data on contaminant-specific biomarkers in fishes is sparse. Traditionally, detection and quantification of heavy metals in sediment, water, and biota gave us valuable information on the quantity and the type of heavy metal present in the ecosystem. This information can be utilized to select a heavy metal specific biomarker. For an instance, if Cadmium (Cd), Zinc (Zn) and Cupper (Cu) are at high concentration, then Metallothionein (MT) can be a good candidate biomarker. Along with this, Superoxide dismutase (SOD) is a very potent indicator of Iron (Fe) and Mercury (Hg) contamination and also Catalase (CAT) is specific for Cadmium (Cd) and Zinc (Zn) exposure. For these kinds of selection of biomarker, the researchers should know heavy metals type specific biomarker. This review is the small effort towards cumulating the heavy metals type specific biomarker. This demonstrates the exposure and effects of heavy metals in fishes by integrating the heavy metal quantification and biomarker selection.
Article
Application of biomarkers for assessing marine environmental health risk is a relatively new field. According to the National Research Council and the World Health Organization, biomarkers can be divided into three classes: biomarkers of exposure, biomarkers of effect, and biomarkers of susceptibility. In order to assess exposure to or effect of the environmental pollutants on marine ecosystem, the following set of biomarkers can be examined: detoxification, oxidative stress, biotransformation products, stress responses, apoptosis, physiological metabolisms, neuromuscular responses, reproductions, steroid hormones, antioxidants, genetic modifications. Since early 1990s, several biomarker research groups have developed health indices of marine organisms to be used for assessing the state of the marine environment. Biomarker indices can be used to interpret data obtained from monitoring biological effects. In this review, we will summarize Health assessment Index, Biomarker Index, Bioeffect Assessment Index and Generalized Linear Model. Measurements of biomarker responses and development of biomarker index in marine organisms from contaminated sites offer great a lot of information, which can be used in environmental monitoring programs, designed for various aspects of ecosystem risk assessment.
Article
Increasing evidence suggests that sublethal effects of natural or xenobiotic chemicals in the environment may be mediated via the stimulation of apoptosis. To investigate whether apoptosis can be induced in fish by weakly estrogenic and androgenic chemicals, adult male Japanese medaka (Oryzias latipes) were exposed to 100 ppb of the estrogenic alkylphenol, 4-nonylphenol, and adult female medaka were exposed to 100 ppb of the aromatase-inhibiting bioflavonoid, quercetin, for 6 weeks. Exposure to nonylphenol and quercetin had no significant effect on the length, weight or condition factors compared to solvent (acetone) controls in male or female medaka. Apoptosis was evaluated in blinded histological sections of whole medaka using terminal dideoxynucleotidyl transferase dUTP nick end labeling (TUNEL) that labels nuclei of cells containing apoptotic (fragmented) DNA. There was a six-fold greater extent of apoptosis in spermatocytes, Sertoli cells and Leydig-homologue cells, but not in spermatids of testes from nonylphenol-exposed male medaka compared to testes of solvent controls. No significant differences in the extent of apoptosis were detected in intestine, liver or kidney from the same male fish. Quercetin-treated female medaka had a significantly increased number of atretic ovarian follicles, but no significant differences in the extent of apoptosis in intestine, liver or kidney. These results suggest that nonylphenol caused testicular degeneration via increased testicular cell apoptosis, while quercetin may be ovotoxic via increased follicular atresia.
Article
We prove here that serum albumin inhibits apoptosis induced by polychlorinated biphenyls (PCBs), confirming that serum albumin binds to PCB, and that the albumin-PCB complexes inhibit apoptosis in HL-60 cells. We found that PCB (50 microM) increased the activity of caspase-3-like protease when HL-60 cells, as well as splenocytes, were cultured in "serum-free medium." Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited apoptosis in cells cultured in the serum-free medium containing 50 microM PCB. To elucidate whether or not PCBs induce apoptosis in vivo, we examined apoptosis of splenocytes by administering PCB to ICR mice (100, 500, 1000 mg x kg(-1) x d(-1)) for 5 d and characterizing splenocytes. Interestingly, splenocytes treated with PCB did not show any changes characteristic of apoptosis. These results demonstrate that PCB activates the caspase-3-like death protease in vitro in serum-free medium, but does not induce apoptosis of splenocytes in vivo, suggesting that blood serum may mask the apoptosis induced by PCB.
Article
Much progress has been made in abating the impacts on aquatic ecosystems of industrial wastewaters, intensive agriculture, and large urban centres. Nowadays the short term consequence of stress from such sources is less frequently the death and destruction of fish populations or entire communities of organisms. Large scale fish kills are now less common. Scientific attention has shifted to the effects on ecosystems of long term exposures to sublethal stressors. Although use of the term ‘ecosystem health’ is a topic of debate, the metaphor can usefully reflect a state of well being or absence of impaired survival, growth, reproduction, and recruitment problems in an ecosystem's key organisms. The present article explores the use of the physiological and biochemical responses of organisms to stressors, the so-called ‘biomarkers,’ to assess and study the sublethal effects of chemical stressors in fish. Fish were chosen as the organism of example because they are key components of practically all aquatic ecosystems. Most biomarkers can be used as an early warning that fish have been exposed to putative stressors, and can often be used to help identify the stressor(s). Biomarkers, however, tell us little about the eventual ecological outcome of such exposures. Some biomarkers are mechanistically linked to toxic modes of action, and can thus be classed as ‘biomarkers of effect’ at the level of the individual organism. None, however, have been fully validated and calibrated as predictive indicators of adverse ecological effects at either the population or community levels. Biomarkers are valueable as part of a broader strategy for monitoring the effects of stressors on aquatic ecosystems.
Article
Atlantic salmon parr were reared for 4 months on experimental diets supplemented with 0 (control), 0.5, 5, 25, 125, or 250 mg Cd x kg(-1) feed to establish a threshold concentration for dietary cadmium exposure by assessing early adaptive cellular responses. At the end of the experiment, the lowest dietary Cd concentration that caused significant accumulation in the gut, kidney and muscle was 5 mg Cd x kg(-1) compared to the control group. Over time, dietary Cd accumulated first in the gut (after 1 month), followed by the kidney (2 months), and later by muscle (4 months). Highest Cd accumulation (100-fold) was found in the gut. A significant increase in regulated cell death and proliferation in salmon fed 125 mg Cd x kg(-1) compared to control fish appeared efficient in preventing gross histopathological damage in the intestine. The highest increase in metallothionein levels was found in the kidney, and metallothionein (MT) levels increased disproportionally to Cd accumulation at increased exposure concentrations. It was concluded that MT was not directly associated with long-term Cd accumulation. Atlantic salmon showed increased metallothionein levels in the kidney at a median effective concentration (concentration of dietary Cd giving 50% of the maximum increase in metallothionein, EC50) of 7 mg Cd x kg(-1), indicating toxic exposure at this concentration.
Article
17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives and hormone replacement therapy, tamoxifen (Tmx), a selective estrogen-receptor modulator used in hormone replacement therapy, and G1, a G protein-coupled estrogen receptor (GPER) selective agonist, differentially increased the hepatic vitellogenin (vtg) gene expression and altered the immune response in adult gilthead seabream (Sparus aurata L.) males. However, no information exists on the effects of these compounds on the immune response of juveniles. This study aims, for the first time, to investigate the effects of the dietary intake of EE2, Tmx or G1 on the immune response of gilthead seabream juveniles and the capacity of the immune system of the specimens to recover its functionality after ceasing exposures (recovery period). The specimens were immunized with hemocyanin in the presence of aluminium adjuvant 1 (group A) or 120 (group B) days after the treatments ceased (dpt). The results indicate that EE2 and Tmx, but not G1, differentially promoted a transient alteration in hepatic vtg gene expression. Although all three compounds did not affect the production of reactive oxygen intermediates, they inhibited the induction of interleukin-1β (il1b) gene expression after priming. Interestingly, although Tmx increased the percentage of IgM-positive cells in both head kidney and spleen during the recovery period, the antibody response of vaccinated fish varied depending on the compound used and when the immunization was administered. Taken together, our results suggest that these compounds differentially alter the capacity of fish to respond to infection during ontogeny and, more interestingly, that the adaptive immune response remained altered to an extent that depends on the compound.
Article
In the present study, the intensity of degenerative changes (apoptosis, necrosis) in the cells of the midgut glands of male and female wolf spiders, Xerolycosa nemoralis (Lycosidae), exposed to natural (starvation) and anthropogenic (the organophosphorous pesticide dimethoate) stressors under laboratory conditions were compared. The spiders were collected from two differentially polluted sites, both located in southern Poland: Katowice–Welnowiec, which is heavily polluted with metals, and Pilica, the reference site. Starvation and dimethoate treatment resulted in enhancement of apoptotic and necrotic changes in the midgut glands of the spiders. The frequency of degenerative changes in starving individuals was twice as high as in the specimens intoxicated with dimethoate. The percentage of apoptotic and necrotic cells was higher in starving males than in starving females. A high intensity of necrotic changes, together with increased Cas-3 like activity and a greater percentage of cells with depolarized mitochondria, were typical of starving males from the polluted site. The cell death indices observed in females depended more strongly on the type of stressor than on previous preexposure to pollutants.
Article
The present study was undertaken to investigate the influence of natural and anthropogenic stressors on the induction of apoptosis, metallothionein (MT) isoforms, heat shock proteins and DNA strand breaks in the marine flatfish dab (Limbanda limanda) Seasonal changes and possible physiological influences were evaluated over a 1-year period at a fixed location northwest of Helgoland in the German Bight. These results were compared with data from sampling sites in the North Sea and the Baltic Sea. Annual cycles could be observed for all parameters except for Cd. The data revealed that changes in biomarker are not only linked to physiological processes related to reproduction but also to factors like water temperature changes, lipid content and zinc content. Cd and organochlorines had no influence on biomarkers whereas an influence of Cd on MT levels revealed in the regional observations was possibly masked by the major changes in Zn content during the annual cycle. Due to different abiotic factors we supposed that the annual cycles at each sampling site in the North and the Baltic Sea might be shifted temporally and therefore measurements at different locations during a small time window of a few weeks may lead to misinterpretation in biomarker research.
Article
Atlantic salmon Salmo salar were fed fish feeds based on 100% replacement of fish oil (FO) with plant oil (PO) as compared to a 100% FO-based diet. The transcript levels of eight genes’ encoding proteins involved in the cellular response to stressors [metallothionein-A isoform (MT-A), cytochrome P450 1A (CYP1A), heat-shock protein 70 (HSP70), catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR), proliferating cell nuclear antigen (PCNA) and caspase 6A] were quantified and compared to cell division (% PCNA-positive cells) and apoptosis [% terminal transferase mediated dUTP nick end labelling technique (TUNEL)-positive cells] in the mid intestinal (MI) section of the gastrointestinal tract. Five of these genes were also quantified in the pylorus caeca region (PR). Fish fed 100% PO-based diets had significantly lower density of both PCNA and TUNEL-positive cells in the MI section compared to fish fed a 100% FO-based diet. The transcription levels of GST and caspase 6A were found to be significantly lower in the MI section of individuals fed a PO-based diet compared to a FO-based diet, analysed by a t-test. In the PR, GR expression was significantly lower in the PO group compared to the FO group. The apoptosis markers PCNA and TUNEL were lower in the group fed a PO-based diet. The results suggest that the transcription levels of three of the studied genes, GST, caspase 6A and GR, can be used as indicators of the MI response to feeding S. salar a PO-based diet.
Article
This chapter reviews the recent developments in the molecular and biochemical studies of metazoan cell death and summarizes recent applications of this new information to the studies of toxicology in fishes. Cell death (apoptosis) plays an important role in the development and function of tissues and organs in all multicellular organisms, including fishes. Cell death can be induced by specific developmental cues that promote active, targeted cell removal (programmed cell death) or can be the passive consequence of pathological or toxicological cell injury (accidental cell death). The chapter explains apoptosis, apoptosis as a cellular response to toxicant exposure, and cell-death detection. Apoptosis is an active, gene-directed program of cell death that results in the deliberate self-destruction and orderly removal of individual cells from a tissue. This suicidal pathway to cell death plays an instrumental role in many biological processes, including tissue morphogenesis and homeostasis, nervous system development, immune system function, and germ-cell selection. There is abundant and accumulating evidence to suggest that apoptosis may be a biologically significant response to toxicant exposure. The apoptotic removal of cells whose function has been compromised by toxicant exposure is one means by which an organism can minimize the deleterious effects of toxicant exposure. A variety of approaches are routinely used to qualitatively or quantitatively assess toxicant-induced cell death in vitro.
Article
Complex environmental mixtures such as pulp mill effluents and crude oil have been shown to increase ovarian cell apoptosis and affect heat shock protein (HSP) expression in fish. We hypothesize that polyaromatic hydrocarbons (PAH) mediate these effects. To test this hypothesis, we exposed juvenile channel catfish (Ictalurus punctatus) acutely to the aryl hydrocarbon receptor (AhR) agonists, beta-naphthoflavone (BNF; 75 mg/kg) or the model PAH, dimethylbenz[a]anthracene (DMBA; 50 mg/kg) via intraperitoneal injection. Apoptotic DNA fragmentation and HSP70 expression were determined in ovary and liver. Hepatic cytochrome P450 1A (CYP1A) was significantly induced, confirming that BNF and DMBA had distributed to internal organs and stimulated AhR. At 96 h post-injection, BNF and DMBA significantly increased apoptosis and decreased HSP70 expression in juvenile catfish ovaries. Although primary oocytes underwent the greatest rates of apoptosis compared to early or late vitellogenic follicles in all treatment groups, the cell type undergoing increased rates of apoptosis after BNF or DMBA exposure was not clear using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyUTP nick end labeling (TUNEL). There was a significant negative relationship between expression of HSP70 and apoptosis in juvenile channel catfish ovaries. This differed from liver of these fish which did not exhibit increased apoptosis and instead increased hepatic HSP70 expression at 96 h post-injection. However, DMBA had no effect on apoptosis or HSP70 levels in more reproductively mature juvenile fish that were housed at a lower water temperature. This may be due to a developmental or temperature-dependent component to these responses. We propose that the decrease in ovarian HSP70 expression in response to BNF and DMBA may be causally related to the increase in ovarian cell apoptosis. Further experiments using a full time course, dose-response and methods to confirm that AhR is a direct mediator of these effects are required.
Article
Full-text available
Apoptosis is a special form of cell death in which the cells actively participate in the process of dying. We found that the water pollutants tributyltin (> 1 muM) and methyl mercury (MeHg; at concentrations of > 3 muM) induce apoptosis in human T-lymphoblastoid CEM cells and in tissue of the marine sponge Geodia cydonium. At concentrations of > 5 muM, MeHg causes alkaline-labile sites in DNA of CEM cells. At the lower dose of 0.3 muM, MeHg abolishes the tributyltin-induced apoptosis in both CEM cells and sponge tissue. Incubation of sponge tissue with 3 muM of tributyltin induces not only apoptosis but also an increased expression of heat shock protein-70.
Article
Full-text available
Cell death by apoptosis mediates several important physiologic and pathologic processes and appears to be intrinsically programmed. Its characteristic features are distinctive morphologic changes of nucleus and cytoplasm, along with cleavage of chromatin at regularly spaced sites. Here we study DNA organization and nuclear structure in apoptotic thymocytes to define the cleavage event and, by implication, the role of the responsible endonuclease. We show that in apoptosis, double-stranded cleavage of DNA generates two classes of chromatin fragments: 70% of DNA exists as long, H1-rich oligonucleosomes bound to the nucleus, and 30% comprises short oligonucleosomes and mononucleosomes, which are depleted in H1, enriched in HMG1 and 2, and not attached to the nucleus. This minority class probably derives from chromatin in a transcriptionally active configuration, which would allow better access to enzymes in the nucleoplasm, producing more complete digestion. The characteristic nucleolar morphology in apoptosis can also be explained in terms of cleavage of the transcriptionally active ribosomal genes, with conservation of the nucleolin-rich fibrillar center. The chromatin cleavage, nucleolar morphologic changes, and chromatin condensation were closely mimicked by micrococcal nuclease digestion of normal thymocyte nuclei in the presence of protease inhibitors. Thus, in apoptosis, selective activation of an endogenous endonuclease appears to be responsible not only for widespread chromatin cleavage but also for the major nuclear morphologic changes.
Article
Full-text available
Copper at low doses is known to specifically induce olfactory neuron death in fish olfactory epithelium. Using light and electron transmission microscopy, we have investigated the features and the time-course of receptor cell death in rainbow trout exposed for 15 days to 20 mug Cu(2+)/l. Ultrastructural observations demonstrate that degenerating cells, which included both mature and immature neurons, exhibited morphological changes characteristic of a cell death by apoptosis. Quantitative analysis shows that the number of apoptotic cells increased significantly already after 1 day of exposure, reaching a peak at day 5. From this timepoint of exposure, no more mature neuron was noted in the olfactory epithelium. Following a significant decrease in the number of apoptotic cells at day 10, a second wave of neuron death was noted at day 15. These findings argue for the occurrence of a neurogenesis process to balance the receptor cell death, despite continued copper exposure, and for a higher vulnerability to the metal of olfactory neurons presenting more advanced stages of cell differentiation. The molecular mechanisms by which copper may induce olfactory neuron apoptosis are discussed.
Article
In near-physiological concentrations, glucocorticoid hormones cause the death of several types of normal and neoplastic lymphoid cell, but the mechanisms involved are unknown. One of the earliest structural changes in the dying cell is widespread chromatin condensation, of the type characteristic of apoptosis, the mode of death frequently observed where cell deletion seems to be 'programmed'. It is shown here that this morphological change is closely associated with excision of nucleosome chains from nuclear chromatin, apparently through activation of an intracellular, but non-lysosomal, endonuclease.
Article
The content of five cyclic organochlorine compounds was determined in eggs of Pagurus bernhardus taken from the North Sea during two seasons. The seasonal pattern of the 24 PCB congeners is influenced by the uptake of food from the spring plankton bloom in early summer and the lipid reserves from the hepatopancreas in winter. Eggs show higher concentrations of ∑ PCB, p, p’-DDE, α-HCH, Lindane, and HCB than abdomens, based on the analysis of n-hexane extractable lipids; no correlation was on the basis of dry weight.
Article
Two liquid Chromatographic stationary phases, dinitroanilinopropylsilica (DNAP) and tetranitrofluorenimino-propylsilica (TENF), are demonstrated to retain selectively the toxic non-ortho-chlorobiphenyl congeners CB-77, CB-126 and CB-169. On the DNAP column, these three congeners elute as a single peak. With hexane as mobile phase all eighteen tested chlorobiphenyl congeners are eluted within 13 min. Carryover effects of CBs in HPLC systems are discussed. To demonstrate the isolation of the three congeners, a sample of Aroclor 1254 was fractionated and analysed by GC with electron-capture detection.
Gamma-irradiation or introduction of hydrocortisone bring about degradation of nuclear DNA in rat thymocytes. The chromatin degradation products were extracted from purified nuclei by 0.7 mM EDTA. The quantity of low molecular weight chromatin fragments formed 6 h after irradiation increases up to the doses of 3 Gy, then remains constant up to 30 Gy and decreases at doses 100 to 300 Gy. Whatever the irradiation dose, DNA degradation starts after a 2-h lag, reaches a maximum by the 6th hour and remains constant between the 6th and 10th hours. The quantity of chromatin fragments formed coincides with the number of cells with pycnotic nuclei. The chromatin fragments present nucleosomes and their oligomers with a normal histone content and an intact structure, as judged from how they are split by DNAase I. The number of intranucleosomal breaks in DNA is negligible. DNA fragmentation is not accompanied by degradation of histones and nonhistone proteins of chromatin. Hence, DNAase I and proteases are not involved in degradation of chromatin. The ratio between mononucleosomes and oligomeres of different lengths does not depend on the dose and the time after irradiation. The quantity of DNA degraded is determined by the number of dying cells in which all DNA is fragmented rather than the degree of chromatin degradation over the whole thymocyte population. Hydrocortisone-induced degradation of chromatin in rat thymocytes occurs similarly. A possible role of chromatin degradation in cell death is discussed.
Article
DNA strand breaks in seastars and dab were measured by the time-dependent partial alkaline unwinding of DNA followed by the determination of the double-stranded to total DNA ratio (F-value). Highest DNA integrity (0.75 < F < 0.85) was found in seastars from offshore reference sites, whereas lowest integrity (0.35 < F < 0.55) was identified in specimens from the coastal zone and certain expected uncontaminated offshore areas. A significant correlation existed between the fraction of double-stranded DNA and the concentration of low chlorinated biphenyl congeners and with H atoms substituted in the meta and para position of the biphenyl skeleton. Over 90% of double-stranded DNA was measured in dab obtained from pristine areas, with an average F-value in specimens from most sampling stations varying from 0.75 to 0.85, during August/September 1991. Samples taken in May/June 1992 showed significantly lower DNA integrity (0.55 < F < 0.70 in most stations). A significant decrease of the integrity was established with increase in concentration of the congeners (tri to hepta chloro substituted and with or without a H atom substituted in the meta and para position).
Article
Concentrations of the non-ortho substituted (planar) chlorobiphenyls 77, 126 and 169, the mono-ortho substituted chlorobiphenyls 105, 118 and 156 and chlorinated dibenzo-p-dioxins and dibenzofurans have been determined in marine and freshwater fish and shellfish from the Netherlands. There are strong indications for a metabolic degradation of the CBs 77 and 126 in yellow eel (Anguilla anguilla). In all fish and shellfish samples analyzed except in yellow eel the toxic effect of the PCBs which in Dutch fish and shellfish is about 4 times higher than that of chlorinated dibenzo-p-dioxins and dibenzofurans, is caused for 85–90% by the CBs 126 and 77. In yellow eel the toxic effect of the PCB is caused for 85–90% by the CBs 126, 156 and 118. The correlation between the CB 153 concentrations and TCDD-equivalents calculated for PCBs enables a prediction of the TCDD-equivalents from the CB 153 concentrations.
Article
Cadmium, a potent toxic metal, poses a serious environmental threat but the mechanisms of its toxicity remain unclear. In the present study, we investigated the nature of cadmium-induced cell death in the human T cell line CEM-C12. Cadmium was time- and dose-dependently toxic for CEM-C12 cells, cell death being preceded by chromatin condensation and DNA fragmentation. Quantification of the latter indicated an increase above 4 μM cadmium, with maximal fragmentation at 8 to 10 μM. By contrast, when CEM-C12 cells were exposed to higher cadmium concentrations (50 μM), cell death increased without concomitant chromatin condensation or DNA fragmentation. Thus, cadmium at low and high concentration kills CEM-C12 cells by apoptosis and necrosis, respectively. Addition of cycloheximide reduced the apoptotic effect of cadmium, suggesting that cadmium-induced apoptosis is an process depending on protein synthesis. Verapamil, a calcium/potassium channel blocker, markedly increased the viability of CEM-C12 cells treated by low cadmium concentrations and prevented DNA fragmentation. The apoptotic effect of cadmium suggests a possible mechanism for lymphocyte damage occuring after in vivo exposure to cadmium.
Article
The effects of intraperitoneal administration of varying doses of cadmium on hepatic metal and xenobiotic detoxication systems in the plaice, Pleuronectes platessa, were studied. The results showed that above a threshold of ca. 2 μg Cd/g liver, metallothionein (MT) levels were increased but at high doses the sequestration capacity of induced MT was exceeded and at the highest dose tested (1 mg Cd/kg) MT induction/synthesis was reduced and hepatic Zn levels decreased. Cadmium injection strongly reduced cytochrome P-450 dependent ethoxyresorufin o-deethylase (EROD) activity and preliminary immunological studies indicated that this was due to a decrease in enzyme protein rather than direct inhibition of activity by Cd. At the sampling time of this study (6 days) there was no significant alteration in activity of the phase II enzyme, glutathione-s-transferase.
Article
As a model for the recognition of effete cells by their viable neighbours. BALB/c mouse thymocytes were coincubated with isologous peritoneal macrophages. The macrophages bound preferentially to thymocytes undergoing apoptosis, a mode of death induced in these cells by treatment with the glucocorticoid hormone methyl-prednisolone. Binding occurred in the absence of serum and was inhibited by N,N'-diacetyl chitobiose, N-acetyl glucosamine and, to a lesser extent, by N-acetyl galactosamine and D-galactose. L-fucose, D-mannose and N-acetyl neuraminic acid had no effect. The results suggest the presence of lectin-like molecules on the surface of the macrophage that recognize changes in the cell-surface carbohydrate of the apoptotic cell. The pattern of inhibition of binding by monosaccharides differs from that of previously described endogenous mammalian lectins.
Article
The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations. Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological. The structural changes take place in two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments. In the second stage these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells. Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development. It occurs spontaneously in untreated malignant neoplasms, and participates in at least some types of therapeutically induced tumour regression. It is implicated in both physiological involution and atrophy of various tissues and organs. It can also be triggered by noxious agents, both in the embryo and adult animal. ImagesFig. 8-10Fig. 1Fig. 2Fig. 3Fig. 4Fig. 6Fig. 7Fig. 11-14Fig. 15-18Fig. 19Fig. 20-22Fig. 23 and 24
Article
In near-physiological concentrations, glucocorticoid hormones cause the death of several types of normal and neoplastic lymphoid cell, but the mechanisms involved are unknown. One of the earliest structural changes in the dying cell is widespread chromatin condensation, of the type characteristic of apoptosis, the mode of death frequently observed where cell deletion seems to be 'programmed'. It is shown here that this morphological change is closely associated with excision of nucleosome chains from nuclear chromatin, apparently through activation of an intracellular, but non-lysosomal, endonuclease.
Article
The classification of cell death can be based on morphological or biochemical criteria or on the circumstances of its occurrence. Currently, irreversible structural alteration provides the only unequivocal evidence of death; biochemical indicators of cell death that are universally applicable have to be precisely defined and studies of cell function or of reproductive capacity do not necessarily differentiate between death and dormant states from which recovery may be possible. It has also proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances. One of these patterns is the swelling proceeding to rupture of plasma and organelle membranes and dissolution of organized structure—termed “coagulative necrosis.” It results from injury by agents, such as toxins and ischemia, affects cells in groups rather than singly, and evokes exudative inflammation when it develops in vivo. The other morphological pattern is characterized by condensation of the cell with maintenance of organelle integrity and the formation of surface protuberances that separate as membrane-bounded globules; in tissues, these are phagocytosed and digested by resident cells, there being no associated inflammation.
Article
Gamma-irradiation or introduction of hydrocortisone bring about degradation of nuclear DNA in rat thymocytes. The chromatin degradation products were extracted from purified nuclei by 0.7 mM EDTA. The quantity of low molecular weight chromatin fragments formed 6 h after irradiation increases up to the doses of 3 Gy, then remains constant up to 30 Gy and decreases at doses 100 to 300 Gy. Whatever the irradiation dose, DNA degradation starts after a 2-h lag, reaches a maximum by the 6th hour and remains constant between the 6th and 10th hours. The quantity of chromatin fragments formed coincides with the number of cells with pycnotic nuclei. The chromatin fragments present nucleosomes and their oligomers with a normal histone content and an intact structure, as judged from how they are split by DNAase I. The number of intranucleosomal breaks in DNA is negligible. DNA fragmentation is not accompanied by degradation of histones and nonhistone proteins of chromatin. Hence, DNAase I and proteases are not involved in degradation of chromatin. The ratio between mononucleosomes and oligomers of different lengths does not depend on the dose and the time after irradiation. The quantity of DNA degraded is determined by the number of dying cells in which all DNA is fragmented rather than the degree of chromatin degradation over the whole thymocyte population. Hydrocortisone-induced degradation of chromatin in rat thymocytes occurs similarly. A possible role of chromatin degradation in cell death is discussed.
Article
A new method is described which is suitable for reliably analysing apoptotic fragmentation in small amounts of DNA. After isolation, DNA was labelled with biotin-4-dUTP using Klenow polymerase. Then DNA was size-separated by agarose gel electrophoresis, blot transferred and subsequently visualized by the streptavidin alkaline phosphatase-BCIP/NBT procedure. This non-radioactive method was used to detect apoptotic DNA in rat pheochromocytoma PC12 cells, treated with tributyltin (1 nM). While only 30 ng of DNA is required for analysis of apoptotic DNA using the new blot technique, 100-fold more material is needed to identify the fragmentation of DNA after separation by agarose gel electrophoresis and direct staining with ethidium bromide. In a further set of experiments, rat cortical cells were incubated with human immunodeficiency virus type 1 viral glycoprotein of M(r) of 120 kDa (gp120) to induce apoptosis. More than 0.3 ng of gp 120/ml are required to detect apoptotic DNA by the direct procedure; only 0.1 ng gp120/ml or less were sufficient to document clear DNA fragmentation using the non-radioactive blotting technique described here. These results demonstrate that the new procedure can be used to analyse very small amounts of apoptotic DNA and shows that gp120-induced apoptosis can be measured at low concentrations of the viral protein.
Article
A number of enzymatic techniques have recently been developed to detect DNA fragmentation in apoptosis at the cellular level. However, since DNA fragmentation also occurs in cellular necrosis, we studied to which extent the use of DNA polymerase (nick translation) or terminal transferase (tailing) allows the differentiation between internucleosomal DNA degradation, typical for apoptosis, and the more random DNA destruction in necrosis. We compared these techniques on in vitro and in vivo models for apoptotic or necrotic cell death. Apoptosis of thymocytes in vitro was induced by gamma-irradiation, necrosis by the cytotoxic action of antibody and complement. Cell death in vivo was examined on paraffin-embedded tissue material from animals with autoimmune encephalomyelitis that served as a model for apoptosis, or in kainic acid-induced nerve cell degeneration as a model for necrosis. DNA fragmentation was visualized by the incorporation of labeled nucleotides into the nuclei of affected cells utilizing tailing or nick translation techniques. In the early stages of cell degeneration in vitro, cells undergoing apoptosis were preferentially labeled by tailing, whereas necrotic cells were identified by nick translation. Similarly, early stages of necrosis in vivo were preferentially detected by nick translation, whereas tailing was slightly more sensitive for the detection of apoptosis. Results obtained with these enzymatic techniques were in accord with the assessment of cell death by morphologic criteria. Both techniques could be applied in tissue samples even after prolonged fixation in paraformaldehyde if the sections were pretreated with proteinase K digestion. Our studies show that both in situ nick translation and in situ tailing are useful in detecting DNA fragmentation in cell suspensions and tissue sections. These techniques may help to define the molecular mechanisms leading to cell death in experimental conditions and eventually in human tissue.
Article
Apoptosis is a form of cell death defined by morphological and biochemical characteristics. Although originally described in 1972, it is only very recently that significant interest in the subject has occurred, possibly as a result of the identification of genes that may either positively or negatively regulate the process. With the rapid expansion of knowledge, it has become apparent that there are multiple pathways that induce apoptosis; some of these may represent programmed events but others are clearly unprogrammed. To clarify the terminology used, it is recommended that apoptosis be used as originally defined to refer only to the end product of these pathways. Furthermore, the realization that a cell can die by multiple pathways suggests caution when translating experimental results. Many potential intermediates may be identified but they may represent components of different pathways. Concern for the use of various inhibitors of apoptosis is also presented. Future directions will be aimed at the definitive identification of the signal mechanisms regulating apoptotic cell death.
Article
A method combining the advantages of electrophoretic DNA fractionation and autoradiography is described for the qualitative and quantitative analysis of internucleosomal DNA fragmentation that occurs during apoptosis, or "programmed cell death." This procedure utilizes terminal transferase enzyme to uniformly add one molecule of [alpha 32P]-dideoxynucleotide to the 3'-end of DNA fragments. Following gel electrophoresis and autoradiographic analysis, the total amount of radiolabel incorporated into the low molecular weight DNA fraction can be quantitated and used to estimate the degree of apoptotic DNA fragmentation in any given sample. This method requires as little as 15 ng of total cellular DNA and increases the sensitivity of apoptotic DNA detection by at least 100-fold over the widely used ethidium bromide staining method. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells.
Article
In vivo CdCl2-induced apoptotic DNA fragmentation in the testes of the male Wistar rat has been demonstrated on agarose gel. Characteristic DNA migration patterns (laddering) provide evidence of apoptosis (programmed cell death) in testicular tissue of rats administered CdCl2 at a level of 0.03 mmol/kg 48 h previously. Evidence that administration of an appropriate cadmium chelating agent within the first 24 h can suppress some or all of the apoptotic changes in testicular DNA has also been obtained for the first time. A greater reduction in apoptosis is observed as the interval between the administration of the cadmium and that of the chelating agent is shortened. Administration of monoisoamyl meso-2,3-dimercaptosuccinate (Mi-ADMS) to male Wistar rats given CdCl2 is effective in the modulation of the typically apoptotic DNA fragmentation and associated histopathologic injury when the antagonist is given within approximately 1 h after the CdCl2 exposure. When the antagonist is given at later times there is a progressively more pronounced degradation of the DNA into oligonucleotides as seen in the typical electrophoretic DNA ladder pattern found with apoptosis. There is also a progressive increase in histopathological tissue changes as the antagonist is administered at progressively greater intervals after the cadmium.
Apoptosis: A product of programmed and unprogram-med cell death Cadmium induces apoptosis in a human T cell line DNA integrity as a biomarker of marine pollution: Strand breaks in seastar
  • A B Eastman
  • G Tsangaris
  • Th
  • O Pellegrini
  • Y Manuel
  • J Benveniste
  • Y Thomas
Eastman, A. (1993). Apoptosis: A product of programmed and unprogram-med cell death. ¹oxicol. Appl. Pharmacol. 121, 160—164. El Azzouzi, B., Tsangaris, G. Th., Pellegrini, O., Manuel, Y., Benveniste, J., and Thomas, Y. (1994). Cadmium induces apoptosis in a human T cell line. ¹oxicology 88, 127—139. Everaarts, J. M. (1995). DNA integrity as a biomarker of marine pollution: Strand breaks in seastar (Asterias rubens) and dab (¸imanda limanda).
Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques
  • R Gold
  • M Schmied
  • G Giegerich
  • H Breitschopf
  • H P Hartung
  • K V Toyka
  • H Lassmann
Gold, R., Schmied, M., Giegerich, G., Breitschopf, H., Hartung, H. P., Toyka, K. V., and Lassmann, H. (1994). Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques.¸ab. Invest. 71, 219-225.
Report of an ICES Sea-going Workshop held on RV U/F Argos
ICES (1989). Methodology of Fish Disease Surveys. Report of an ICES Sea-going Workshop held on RV U/F Argos, 16-32 April 1988. ICES Cooperative Research Report 166.
In vivo DNA degradation in thymocytes ofγ
  • Umansky
Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques
  • Gold
Fractionation of non-ortho
  • Grimvall