Environmental Toxicology and Pharmacology

Published by Elsevier
Print ISSN: 1382-6689
Cadmium is a well-known toxic compound for the liver. It has been demonstrated to induce hepatotoxicity partly via apoptosis, but no uniform mechanism of apoptosis has so far been proposed. This study was first to determine whether cadmium-induced apoptosis in L-02 cells, second to observe the mechanism of cadmium-induced apoptosis. Studies of morphology, DNA fragmentation and apoptotic rate demonstrated that 60μM cadmium induced apoptosis with strong effects on cell viability. A concomitant time-dependent decrease of Bcl-2 and mitochondrial transmembrane potential (ΔΨ(m)) was observed. Subsequently, increase of caspase-3 activity and release of mitochondrial AIF were detected. However, cell pretreatment with a broad-specificity caspase inhibitor (Z-Asp) did not abolish apoptosis. These data demonstrated that the apoptotic events involved a mitochondria-mediated apoptotic pathway but not necessarily caspase-dependent signaling. On the other hand, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of cadmium-exposed cells had significant increases and the Bapta-AM, a well-known calcium chelator, pretreatment partially blocked cadmium-induced apoptosis, indicating that the elevation of [Ca(2+)](i) may play an important role in the apoptosis. Together, these results support the notion that cadmium-induced hepatotoxicity is comparable to effects in L-02 by inducing apoptotic pathways on the basis of acting on mitochondria and regulating Ca(2+) signals.
The possible impact of the rocket fuel 1,1-dimethyl hydrazine (heptyl) (1) and its transformation products on human health has been studied using (Quantitative) Structure Activity/Toxicity ((Q)SAR/(Q)STR) modelling, including both ADME models and models for acute toxicity, organ specific adverse haematological effects, the cardiovascular and gastrointestinal systems, the kidneys, the liver and the lungs, as well as a model predicting the biological activity of the compounds. It was predicted that all compounds studied are readily bioavailable through oral intake and that significant amounts of the compounds will be freely available in the systemic circulation. In general, the compounds are not predicted to be acutely toxic apart from hydrogen cyanide, whereas several compounds are predicted to cause adverse organ specific human health effects. Further, several compounds are predicted to exhibit high probabilities for potential carcinogenicity, mutagenicity, teratogenicity and/or embryotoxicity. The compounds were ranked based on their predicted human health impact using partial order ranking methodologies that highlight which compounds on a cumulative basis should receive the major attention, i.e., N-nitroso dimethyl amine, 1,1,4,4-tetramethyl tetrazene, trimethyl, trimethyl hydrazine, acetaldehyde dimethyl hydrazone, 1, 1-formyl 2,2-dimethyl hydrazine and formaldehyde dimethyl hydrazone, respectively.
1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) is the most prevalent metabolite of DDT used as a pesticide before and tributyltin (TBT) compounds are used primarily as antifouling agents on vessels, ships, and aqua culture facilities, as they exert biocidal actions. Currently, p,p'-DDE and TBT are ubiquitously distributed in the environment and bio-accumulated in marine products, especially fish or shellfish. Thus, oral p,p'-DDE and TBT intake through marine products is demonstrated to be rather high in Japan. Consequently, the fetus and neonate will be exposed to p,p'-DDE and TBT via mother. Therefore, effects of perinatal combined exposure to p,p'-DDE and TBT on the female reproductive system after maturation have been investigated in rat female offspring of dams ingesting 125ppm p,p'-DDE (approximately 10mg/kg) and 25ppm TBT (approximately 2mg/kg) during the perinatal period from gestation to lactation. In the present study, no deleterious reproductive outcomes were recognized in p,p'-DDE and/or TBT-treated dams. In contrast, growth retardation had developed in rat female offspring following perinatal exposure to TBT and sustained even after cessation of exposures. Further, reduced ovarian weights with elevated serum follicle-stimulating hormone (FSH) concentrations were observed in the reproductive system of matured female offspring following perinatal exposure to TBT. At present, biological relevance of these alterations remains unknown, but there is a possibility that these alterations lead to reproductive malfunctions in matured female offspring.
The mutagenic effect of the rocket fuel 1,1-dimethyl hydrazine has been studied experimentally and compared to the well-recognized mutagene N-nitroso dimethylamine. The manifestation of the effect for both compounds was disclosed through a significant increase in the chromosome aberration frequency in the bone marrow cells of intoxicated rats. The levels of chromosome aberrations induced by 1,1-dimetyl hydrazine were studied following both single (1h) and repeated doses (daily for 10 consecutive days) by inhalation (205-1028mg/m(3)) and gavage (5.4-26.8mg/kg) administration, respectively. For comparison N-nitroso dimethylamine were administered by inhalation (2h/daily for 10 consecutive days) and by gavage in concentrations of 2.4-48mg/m(3) and 1-30mg/kg, respectively. A clear dependence of concentration as well of time was disclosed. The BenchMark Dose approach was employed to derive guideline doses for the two compounds, the implications towards human health being discussed.
The aliphatic ether 1,6-dimethoxyhexane (DMH) was previously identified as a testicular toxicant. Testis protein extracts from control and DMH-treated rats were subjected to two-dimensional gel electrophoresis for comparison of protein expression profiles. MALDI-ToF peptide mass fingerprinting of differentially expressed proteins resulted in the conclusive identification of heat shock-related 70kDa protein 2 (HSP70.2), 60kDa heat shock protein, mitochondrial precursor (HSP60) and protein disulfide isomerase A3 precursor (ERp60). The potential involvement of these proteins in chemically induced perturbation of spermatogenesis and their utility as biomarkers of testicular toxicity are discussed in light of the knowledge currently available from the literature.
To investigate the role of dopamine D(1) receptors in the reinforcing effects of cocaine and fentanyl, the effect of the D(1) antagonist SCH23390 on intravenous self-administration of these drugs was investigated in rats using a progressive ratio (PR) reinforcement schedule, during which the rats received the first three injections under an FR1 schedule. Then the number of lever presses required to deliver an injection (lever press ratio) increased by three after every three further injections. The last lever press ratio completed by each rat during each 6 h session was designated the breaking point. Breaking point values increased dose-dependently during both cocaine (0.125-1.00 mg/kg per injection) and fentanyl (0.25-2.00 μg/kg per injection) self-administration. Pretreatment with SCH23390 (0.01 mg/kg, s.c.) decreased breaking point values for both cocaine and fentanyl, reflecting a decrease in the reinforcing efficacy of the drugs. To determine whether the effect of SCH23390 was due to general suppression of the lever pressing response, the effect of SCH23390 (0.01 mg/kg, s.c.) on the performance of rats maintained by water-reinforcement was examined. SCH23390 suppressed performance only transiently, therefore general suppression appears to have little or no effect on the breaking point. These results suggest that dopamine D(1) receptors are involved in mediating the reinforcing effects of both the psychostimulant cocaine and the opiate fentanyl.
Monsanto produced two distinct variants of Aroclor 1254. The late-production variant resulted from a change in Monsanto's manufacturing process in the early 1970s. Previous literature had reported that the late-production variant was produced from 1974 to 1976, but subsequent work has identified a sample known to be obtained in 1972. In this paper, we present congener-specific PCB and PCDD/F data for this 1972 late-production sample, and a brief historical record of late-production Aroclor 1254.
This paper focuses on possible activities of children aged 6-13 that may make them susceptible to high hourly intake doses of ozone (O(3)) air pollution. Data from an O(3) exposure modeling exercise indicates that relatively few hours can account for a significant amount of the total cumulative ozone intake dose (D(I)(T)) received in a year. While O(3) concentration levels affected the hourly intake dose (D(Ih)) more than breathing rate (V ̇ (E)), there was a distinct and significant relationship between V ̇ (E) and D(Ih) when hours in a year were stratified by concentration levels. By investigating the V ̇ (E) levels associated with high dose hours, it appears that activities with an hourly V ̇ (E) rate of between 25 and 45 l/min can lead to high intake doses of O(3). The exercise literature was reviewed to ascertain which activities could lead to that range of V ̇ (E)'s and be sustained by children for 1 h. A list of such activities was developed. If an analyst is interested only in modeling high D(Ih) hours for children aged 6-13, an efficient approach might be to focus on those activities and estimate D(I)(T) for the other hours in an aggregated manner.
Grains of soybeans were treated with the recommended dose of fenitrothion insecticide, 5mg/kg, and double that dose, before storage for 30 weeks under ambient local conditions. During the storage period, the penetration and distribution of insecticide residues were studied. The amount of surface residues on stored soybeans, internal extractable and bound residues were determined. Surface residues were found to decrease with the increase in time of storage, whereas internal residues showed a gradual increase with time to reach 56%, 54% of applied doses after 30 weeks. The amount of bound residues inside the matrix showed a slow increase with time. Toxicity of the total internal residues of fenitrothion in stored soybeans was studied in mice through a sub-chronic feeding experiment for 3 months. The maximum inhibition in plasma and erythrocyte cholinesterase activity was 37% and 13% after the first month, respectively. Treated mice suffered from deterioration of hepatic and renal functions as indicated by the increase level of blood serum esterase's and blood urea nitrogen. Percentage increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was reached to about 62% and 55%, respectively as compared with control animals with the end of feeding period.
Estrogenic potency of 17-β estradiol (E2) was evaluated for the first time in both sexually undifferentiated (during resting phase, at the very early stage of gametogenesis) and differentiated (in pre-spawning phase) clams (Tapes philippinarum), following waterborne exposure for 7 and 14 days to various E2 concentrations (0, 0+acetone, 5, 25, 50, 100 and 1000ng/L). Vitellogenin (Vg)-like protein levels were evaluated in both haemolymph and digestive glands by the alkali-labile phosphate (ALP) assay. In the resting phase of clams, exposure for 7 days to all E2 concentrations tested resulted in significant increases in ALP in haemolymph with respect to controls. Conversely, no statistically significant differences were observed in digestive gland from E2-exposed bivalves, compared with controls. After 14-days exposure, ALP significantly increased in haemolymph from 1000ng E2/L-exposed clams and in digestive gland from 5ng E2/L-exposed animals. Interestingly, in the pre-spawning phase of clams, significant decreases in ALP levels were recorded in haemolymph from females exposed for 7 days, whereas no differences were observed in males. No significant variations were observed in ALP content in digestive gland from both females and males. ALP levels in haemolymph were shown to increase significantly in females exposed for 14 days to 50ng E2/L, and in males exposed to 100ng E2/L. Exposure for 14 days to all the E2 concentrations tested resulted in significant increases of ALP levels in the digestive gland of females. Conversely, variations in ALP content of male digestive glands were not significant. Results obtained appeared controversial and somewhat difficult to explain. Indeed, no univocal response by clams was observed after 7 and 14 days exposure to E2. Further studies need to elucidate the role of E2 in promoting Vg induction in clams.
In this study, male Chinese loaches were exposed to 17β-estradiol (E2), bisphenol A (BPA) and their mixtures, respectively, for 42 days using a semi-static exposure system. Plasma vitellogenin (Vtg) in male Chinese loaches was used as the determining endpoint. The results demonstrated that male Chinese loaches were sensitive to E2, and the vitellogenic responses showed time- and dose-dependent increase. Similarly, BPA induced the estrogenic effects in male Chinese loaches, and the vitellogenic response increased in a time- and dose-dependent manner. The synthesis of Vtg was initiated by the exposure to higher level of BPA (500μg/L) within 7 days, and a relative long-term exposure to lower concentrations of BPA (10-100μg/L) also led to the production of Vtg. The estrogenic effect of the binary mixture of E2 and BPA also showed a time- and dose-dependent response, which was more potent than that of individual compounds, and Vtg contents in the binary mixture group were higher than the summation of Vtg contents in the single-compound groups at the same concentration.
Plot adjusted probits and predicted regression line for cyfluthrin to tilapia fry in 48 h.
Acute 48 h toxicity of cyfluthrin to Nile tilapia fry
Plot adjusted probits and predicted regression line for cyfluthrin to tilapia fry in 72 h.
Acute 72 h toxicity of cyfluthrin to Nile tilapia fry
Cyfluthrin, a synthetic pyrethroid pesticide contaminating aquatic ecosystems as a potential toxic pollutant, was investigated in the present study for acute toxicity. Tilapia fry (Oreochromis niloticus L. 1758) was selected for the bioassay experiments. The 48 and 72h LC(50) was determined for the tilapia fry. The experiments were repeated three times. The static test method of acute toxicity test was used. In addition, behavioral changes at each cyfluthrin concentration were determined for the individuals. Data obtained from the cyfluthrin acute toxicity tests were evaluated using the Probit Analysis Statistical Method. The 48 and 72h LC(50) value for tilapia fry were estimated as 25.82μg/L (95% confidence limits: 20.92-43.26) and 21.07μg/L (95% confidence limits: 16.21-30.39), respectively.
The effects of oral administration of tamoxifen, 17α-ethynylestradiol (EE2), flutamide, and methyltestosterone (MT), on plasma vitellogenin levels of male and female medaka were investigated. Medaka were fed diets containing different concentrations of these chemicals for 7 days, and these plasma vitellogenin levels were measured. Tamoxifen increased significantly the vitellogenin levels in male, but inhibited the normal vitellogenin induction in female in the high concentration groups. EE2 increased significantly vitellogenin levels in both sexes. Flutamide increased significantly the vitellogenin levels in female, but gave no effects on male. MT inhibited the normal vitellogenin induction in female, but increased slightly vitellogenin levels in male without a clear tendency. Administration of tamoxifen, EE2, flutamide, and MT showed the different pattern in vitellogenin levels in both sexes.
Effects of 40 days of exposure and 20 days of recovery response at sublethal concentration of technical grades of gamma isomer of hexachlorocyclohexane (γ-HCH, 0.025ppm, 99.8%) and dichlorodiphenyltrichloroethane (DDT, 5.0ppm) in tissue (liver, brain and ovary) bioconcentrations, gonadosomatic index (GSI) and plasma levels of estradiol-17β (E2) have been estimated during prespawning phase in the catfish Heteropneustes fossilis (Bloch). The results indicated that the tissue bioconcentrations of both HCHs (HCH isomers) and DDTs (metabolites of DDT) in liver, brain and ovary were in preferential order (liver>brain>ovary). The GSI and plasma levels of E2 were declined in response to exposure of γ-HCH and DDT. On withdrawal of exposure of pesticide there was recovery of HCHs in exposed fish for all tissues studied, whereas DDTs exposed fish showed recovery only in liver. Recovery of E2 production was also recorded in γ-HCH exposed fish whereas very little recorded in DDT exposed fish. It is suggested that HCHs and DDTs have preferential order (liver>brain>ovary) of their tissue bioconcentrations and HCH/DDT-withdrawal-dependent recovery during studied phase.
The effects of bis(2-ethylhexyl) phthalate (DEHP), γ-hexachlorocyclohexane (γ-HCH), and 17β-estradiol (E2) on the fry stage of medaka were investigated. The medaka fry were exposed to different concentrations (0.01, 0.1, 1, and 10μg/L) of these chemicals for 3 weeks after hatching. Then, mortality, body weight, sex ratio, and gonadosomatic index (GSI) of the matured fish (after 5 months) were measured. Mortality was increased significantly in the 10μg/L E2 group. Distortion of sex ratio was found in 1 and 10μg/L E2 groups. DEHP treated groups showed the GSI reduction only in male fish. All the γ-HCH and parts of the E2 treated groups showed the GSI reduction in both sexes. Exposure of DEHP, γ-HCH, and E2 during the fry stage affected normal maturation of medaka at the concentrations which had no impact on mortality or sex ratio.
Freshwater clams Corbicula fluminea were experimentally exposed to a range of tributyltin (TBT) (50, 250 and 500ng Sn/L) and 17β-estradiol (20, 200, 2000ng/L) for 30 days. After 15 and 30 days, phagocytosis activity of haemocytes and lysosomal structural changes in the digestive cells were assayed. 17β-Estradiol exerted a higher inhibition on phagocytosis than tributyltin. This would suggest the existence of estrogen receptors, influencing the immune function. The stereological parameters measured for lysosomal structural changes in animals exposed to tributyltin varied as observed in other studies. Tributyltin is then depurated as other contaminants via digestive cell lysosomal compartment. This experiment emphasized a possible approach on the influence of endocrine disrupting compounds on a hermaphroditic species for environmental surveys.
There is increasing evidence that health effects of toxic metals, including methylmercury (MeHg), differ in prevalence or are manifested differently in men and women. The present study was aimed at investigating the potential differential susceptibility of male and female Swiss mice against MeHg-induced neurotoxicity, which was evaluated by biochemical (cerebellar oxidative stress-related parameters) and behavioral (locomotor activity and motor performance) variables. We also aimed to evaluate the potential protective effects of 17β-estradiol against such toxicity in MeHg-exposed male animals. MeHg exposure (40mg/L, diluted in tap water, during 2 weeks) decreased locomotor activity and motor performance in both male and female animals, but such phenomena were higher in males. 17β-estradiol co-treatment (10μg/animal, in alternate days) prevented MeHg-induced locomotor deficits in males. MeHg exposure caused a significant increase (60%) in cerebellar lipid peroxidation in male mice, but did not in females. In close agreement, MeHg exposure decreased (43%) cerebellar glutathione peroxidase activity in males, but did not in females. These events were prevented by 17β-estradiol administration. Cerebellar GR activity was increased (25%) in MeHg-exposed males and such event was partially prevented by 17β-estradiol administration. These results indicate that the low susceptibility of female mice to the neurotoxicity elicited by MeHg is linked to neuroprotective effects of sex steroids, which appear to modulate the activities of glutathione-related enzymes. Our experimental observation corroborates previous epidemiological studies showing the greater developmental effects in male than in female humans exposed to MeHg.
Genistein and daidzein receive much attention because of their potential to prevent hormone-related cancer and cardiovascular diseases. Limited information is available on the pharmacokinetics of these compounds like, for instance, intestinal uptake by humans and systematic bioavailability. In this study the transport and metabolism of genistein, daidzein and their glycosides has been compared in various cellular models for intestinal absorption such as human colonic Caco-2, rat small intestinal IEC-18 and human immortalized colon HCEC cell lines. Genistein and daidzein were taken up by Caco-2, IEC-18 and HCEC cells and transported to almost same rate and extents. Glycosides were transported across IEC-18 and HCEC monolayers, but not across Caco-2 cells. In Caco-2 and IEC-18 cells, the glycosides were metabolized to their respective aglycones. Furthermore, it was shown that genistein and daidzein were glucuronidated and sulfated in Caco-2 cells, to glucuronidated forms in IEC-18 cells and to sulfated conjugates in HCEC cells. The results of this study compared with reported in vivo data indicate that Caco-2 cells are a valuable model for studying intestinal transport and metabolism of isoflavones.
Transepithelial permeability of GlySar, methylglucose and mannitol in IEC-18 and Caco-2 cells. Apical to basal transport of 10 m M GlySar, methylglucose (Meth-Gluc) and mannitol was determined from 0 to 240 min in IEC-18 (A) and Caco-2 cells (B). Each point is the mean 9 sd of at least two experiments each performed in triplicate. 
Bidirectional transport of rhodamine 123 in IEC-18 and Caco-2 cells. Apical to basal (Ap Bl) transport and basal to apical (Bl Ap) transport of 20 m M rhodamine 123 was measured in IEC-18 and Caco-2 cells. Each point is the mean 9 sd of at least three experiments each performed in tripicate. 
Calcein ef fl ux from IEC-18 and Caco-2 cells. IEC-18 (A) and Caco-2 (B) cells were loaded for 30 min with 2 m M calcein-AM in presence of 20 m M verapamil and 2 mM probenecid. After transfer of the fi lter-grown cells into prewarmed calcein-free medium, release of calcein to the apical (Ap) and basal (Bl) compartment was measured in absence or presence ( + Prob) of 2 mM probenecid. Each point is mean 9 sd of three independent experiments. 
DNPSG transport and cellular accumulation in IEC-18 and Caco-2 cells. IEC-18 (A) and Caco-2 (B) cells were apically exposed to 25 m M CDNB. Cellular accumulation and transport of DNPSG to the apical and basal compartment was measured during 60 min in the absence or presence of probenecid. Data are mean 9 sd of at least three experiments. 
DNPSG efflux after apical or basolateral exposure of the cells to CDNB
Purpose: previous studies have shown that the rat small intestinal cell line IEC-18 provides a size-selective barrier for paracellularly transported hydrophilic macromolecules. In order to determine the utility of IEC-18 cells as an in vitro model to screen the passive paracellular and transcellular components of the intestinal transport of nutrients and drugs, we have now examined the transport of GlySar (H(+)-coupled di/tripeptide carrier), O-methyl-d-glucose (glucose carrier), vincristine and rhodamine 123 (P-glycoprotein), and calcein and DNPSG (MRPs) and the bidirectional transport of paracellularly transported compounds. Transport of these compounds across the filter grown IEC-18 cells was compared with transport across the human colon carcinoma Caco-2 cells. Results: in IEC-18 cells, transepithelial transport of GlySar and methylglucose was as fast as the transport of mannitol, which is transported passively via the paracellular route. Whereas in Caco-2 cells, mannitol transport was much slower than the transport of GlySar and methylglucose. In contrast to Caco-2 cells, no H(+)-coupled transport of GlySar could be measured in IEC-18 cells. P-Glycoprotein-mediated transport was characterised in Caco-2 cells by an enhanced transport of vincristine and rhodamine 123 in the basolateral to apical direction and by the inhibition of this transport by verapamil. In IEC-18 cells, permeability of vincristine and rhodamine 123 was similar in both directions and verapamil had no effect on the transport of these compounds. Both IEC-18 and Caco-2 cells efflux the organic anions calcein and DNPSG to the apical and basolateral compartments, and this efflux could be inhibited by probenecid. Conclusions: in conclusion, no carrier-mediated transport of GlySar, methylglucose, vincristine and rhodamine 123 could be determined in IEC-18 cells in contrast to Caco-2 cells. However, both IEC-18 and Caco-2 cells showed MRP-mediated eflux system(s) in the apical and basolateral membrane. Monolayers of IEC-18 cells appear to be more suitable than monolayers of Caco-2 cells as an in vitro system to screen the passive component of the intestinal transport in a deconvoluted screening regimen, where passive transport is represented by the IEC-18 monolayer permeability and active transport is represented by monolayers of cells expressing the transport proteins heterologously.
Deltamethrin, an α-cyano type II pyrethroid, administered orally (5, 10 and 15 mg/kg body weight for 7 consecutive days or at 5 mg/kg for further 15 and 21 days) to young albino Wistar rats (approximately 8 weeks old) produced a dose- and time-dependent increase in the activity of cytochrome P450-dependent 7-ethoxyresorufin-O-dealkylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) in rat liver and brain. However, significant induction in the enzyme activities was observed at higher doses or prolonged exposure. The magnitude of induction in rat liver microsomes was less at 15 mg/kg dose as compared to 10 mg/kg dose. Western blot analysis revealed a similar dose-related and time-dependent increase in the expression of P450 2B1/2B2 and 1A1 isoenzymes as indicated by the increased cross-reactivity of liver microsomes isolated from deltamethrin-treated animals with anti-P450 2B1/2B2 and 1A1. Inhibition of EROD and PROD observed after in vitro addition of anti-P450 2B1/2B2 and 1A1/1A2 or organic inhibitors, metyrapone and α-naphthoflavone, to the brain and liver microsomes of deltamethrin-pretreated animals (5 mg/kg×21 days), further provided support that the induction observed in the EROD and PROD activity in brain is due to the increased expression of P450 2B1/2B2 and 1A1/1A2, while, in the liver, isoenzymes other than these are also involved.
Polybrominated diphenyl ethers (PBDEs) are used as flame-retardants and have recently been shown to increase in the environment and in human milk. We have recently reported that neonatal exposure to 2,2',4,4',5-pentaBDE (PBDE 99) can induce persistent aberrations in spontaneous behaviour and also affect learning and memory functions in the adult animal. The present study indicates that the cholinergic system, in its developing stage, can be a target for PBDEs. Neonatal oral exposure of male NMRI mice, on postnatal day 10, to 2,2',4,4',5-pentaBDE (12mg/kg body weight) was shown to decrease the density of cholinergic nicotinic receptors in hippocampus, at an adult age. These findings show similarities to observations made from neonatal exposure to 2,2',4,4',5,5'-hexaBDE, PCBs and nicotine, compounds shown to affect cholinergic nicotinic receptors. The animals of the present study also showed disrupted spontaneous behaviour, and the highest dose that did not cause any significant behavioural disruption was 0.4mg 2,2',4,4',5-pentaBDE/kg b.w.
Polybrominated diphenyl ethers (PBDEs), which are used as flame-retardants, have recently been shown to be increasing in the environment and in human milk. We have recently reported that neonatal exposure to PBDEs, including 2,2',4,4',5-pentaBDE (PBDE 99), can induce persistent aberrations in spontaneous behaviour and also affect learning and memory functions and nicotinic cholinergic receptors in the adult mice. The present study indicates that spontaneous behaviour, along with the cholinergic system during its developing stage, can be targets for PBDEs in the rat as well. Neonatal oral exposure of male Sprague-Dawley rats, on postnatal day 10, to 0.8, 8.0, and 16mg PBDE 99/kg body weight, was shown to disrupt normal spontaneous behaviour in a dose-response related manner at 2 months of age. Also, the animals of the present study showed a decrease in density of muscarinic cholinergic receptors in hippocampus, at an adult age. These findings show similarities to observations made from neonatal exposure of mice to PBDE 99, 2,2',4,4',5,5'-hexaBDE (PBDE 153) and certain PCBs, compounds shown to affect both spontaneous behaviour and the cholinergic system.
The effect of 2,2'-substitution with fluorine, methyl or trifluoromethyl groups on the toxicity, metabolism and pharmacological activity of dapsone has been investigated in vitro and in vivo. There was marked inter-species variation in the bioactivation (N-hydroxylation) of the compounds, as determined by methemoglobin formation. However, the inclusion of fluorine significantly (P<0.01) reduced methemoglobin formation compared with dapsone in all species studied. All three analogs resulted in significantly (P<0.001) less methemoglobinemia than dapsone when given either intraperitoneally or intravenously to the male Wistar rat. Rapid plasma clearance of the analogs through increased lipophilicity and enhanced N-glucuronidation may account for the low toxicity compared with dapsone. Although trifluoromethyl substitution resulted in a loss of activity against respiratory burst in human neutrophils in an in vitro model, all three analogs retained pharmacological activity against Plasmodium berghei malaria in an in vivo mouse model.
After the administration of 2,2',4,5,5'-pentachlorobiphenyl (2,2',4,5,5'-pentaCB) to intact rats, the concentration of 2,2',4,5,5'-pentaCB in liver gradually decreased, whereas 3-methylsulfonyl (3-MeSO(2))-2,2',4',5,5'-pentaCB appeared in liver and remained detectable in liver for 6 weeks. A single injection of 2,2',4,5,5'-pentaCB (342 μmol/kg) or 3-MeSO(2)-2,2',4',5,5'-pentaCB (0.5 μmol/kg) caused a significant increase both in the contents of cytochromes P450 and b(5) and in the activities of aminopyrine N-demethylase and benzo[a]pyrene hydroxylase, and the increased enzyme contents and activities continued for 6 weeks after the administration. The extent of both the hepatic accumulation of 3-MeSO(2)-2,2',4',5,5'-pentaCB and the induction of the enzymes for 6 weeks after the administration of 2,2',4,5,5'-pentaCB was similar to that after the administration of 3-MeSO(2)-2,2',4',5,5'-pentaCB. 3-MeSO(2)-2,2',4',5,5'-pentaCB was considered to play a principal role in the induction of microsomal drug-metabolizing enzymes by 2,2',4,5,5'-pentaCB. When 2,2',4,5,5'-pentaCB was injected i.p. into bile duct-cannulated rats, 3- and 4-MeSO(2)-2,2',4',5,5'-pentaCBs were not detected in liver. In antibiotic-treated rats dosed with 2,2',4,5,5'-pentaCB, the concentrations of 3- and 4-MeSO(2)-2,2',4',5,5'-pentaCBs in liver were markedly reduced. These findings suggest that the process in which 3- and 4-MeSO(2) metabolites of 2,2',4,5,5'-pentaCB are formed involves the biliary secretion of some precursors which will be subjected to metabolism by intestinal microflora. The increasing effects of 2,2',4,5,5'-pentaCB both on the content of cytochrome P450 and on the activity of aminopyrine metabolizing enzyme in hepatic microsomes were not observed in the bile duct-cannulated rats, in which the phenobarbital treatment enabled the drug-metabolizing enzymes to be induced. In antibiotic-treated rats, the increases both in the cytochrome P450 content and in the aminopyrine N-demethylase activity after the administration of 2,2',4,5,5'-pentaCB were smaller than those observed in the intact rats. These findings provide the evidence that the induction of some drug-metabolizing enzymes by 2,2',4,5,5'-pentaCB is due not to the action of 2,2',4,5,5'-pentaCB itself but to its 3-methylsulfonyl metabolite, 3-MeSO(2)-2,2',4',5,5'-pentaCB.
Aromatase catalyzes intraneuronal conversion of androgens to estrogens in the hypothalamus-preoptic area at a specific time during development. This local estrogen formation has a central role in sexual differentiation. To investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on sexual differentiation, TCDD (800 and 1600 ng/kg) was orally administered to pregnant Holtzman rats on gestation day (GD) 15, and the change in brain aromatase activity of fetuses (GD20) and pups (postnatal day 2) was examined. Litter means of brain aromatase activity in the hypo-preoptic area of control fetuses were higher in males than in females (female/male ratio was 0.7). This ratio of aromatase activity increased in a TCDD-concentration dependent manner, reaching 1 at the higher dose. There was a similar change in aromatase activity in brains of pups (PND 2). These results suggest that in utero TCDD exposure induces demasculinization in male offspring by inhibiting the aromatase activity in the brain during central nervous system development.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a powerful toxicant that exerts its effects through the aryl hydrocarbon receptor (AHR) governed by the Ahr locus that in mice is located on chromosome 12. We used single marker analyses of the offspring of female mice treated/not treated with TCDD to search for a gene (quantitative trait locus or QTL) on chromosome 12 near the site of the Ahr locus to test whether this locus appeared to affect mandible size, shape, and/or asymmetry especially in the treated mice. These mice were sampled from the F(2) generation of an original intercross of two strains (C57BL/6J and AKR/J) known to be divergent in their response to TCDD. A QTL affecting mandible shape was found on chromosome 12, but its effect on mice in the treated and control groups did not differ and it was concluded that this QTL probably was not the Ahr locus itself. We also probed a second chromosome (11) and found a QTL whose effects on asymmetry of mandible shape differed in the two environments. These results suggested that the entire genome in these mice should be scanned to search for additional QTLs that might be affected by TCDD to learn more about the potential effects of this powerful toxicant on these genes.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to affect various cellular activities including growth factor signal transduction, hormone responses, and cell differentiation. The purpose of this study was to examine more closely the very early effects of TCDD on protein tyrosine kinase activity, specifically p60(Src). We found that TCDD causes rapid changes in the plasma-microsomal membrane levels and activity of p60(Src) in Hepa 1c1c7, Hepa c4 cells, and SR3Y1 cells, a p60(v-Src) overexpressing cell line. Such cellular changes occur within 30 minutes after 10 nM TCDD treatment, as measured by Western blot analysis. TCDD's ability to increase p60(Src) levels was found to be: (1) dose-dependent, with an estimated EC(50) between 10(-10) and 10(-11) M TCDD; (2) Ah receptor (AhR)-dependent, since TCDD's effect was blocked by co-administration with 1 μM α-naphthoflavone, an AhR antagonist; and interestingly (3) ARNT-independent, since TCDD's effect was observed in Hepa c4 cells, an ARNT(-) mutant cell line. Since ARNT is a heterodimerization partner of the AhR required for binding of the ligand-activated AhR to dioxin-responsive elements on DNA in the nucleus to transactivate genes controlled by the AhR, an alternative mechanism for TCDD's action is discussed which does not require ARNT. Along with increased membrane levels of p60(Src), we observed a corresponding increase in the activity of a 60 kDa protein tyrosine kinase using two different kinase detection assays. This effect of TCDD was also found to be AhR-dependent, ARNT-independent, and independent of de novo protein synthesis since cycloheximide was unable to completely abolish TCDD's effect. The present findings provide a potentially important mechanism by which TCDD can alter cell growth and differentiation.
A PBPK model for 2,4-D was developed that involves flow-limited pH trapping modified to consider tissue binding, binding to plasma, and high-dose inhibition of urinary excretion. The PBPK model provides reasonable estimates of the kinetics of 2,4-D in rats as well as in humans, providing a common metric for expressing risk. The risk characterization for 2,4-D based on the PBPK model is consistent with that based on standard risk assessment methods, except that the apparent variability in the risk characterization is reduced. The model demonstrates that non-linear pharmacokinetics and inhibition of urinary excretion would not be expected in occupational exposures. This case study suggests that preliminary PBPK models could be developed for numerous pesticides based on commonly available data. If properly validated with well-designed worker exposure studies, such models may be useful in more complete assessments of risks to workers as well as members of the general public.
The action of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the isolated heart of the frog (Rana ridibunda) and two insects, the honeybee (Apis mellifera macedonica) and the beetle (Tenebrio molitor), was investigated using basic electrophysiological methods. The results of this study showed that a concentration of 1 μM 2,4-D was required to reduce the force and the frequency of the isolated heart of the honeybee to about 70% of the initial contraction in less than 20 min. To cause the same effects on the atria of the frog, 45 μM 2,4-D was required and on the isolated heart of the beetle, over 1000 μM of 2,4-D. The presence of an extensive system of gap junctions found in the honeybee is most probably the cause of the unusual sensitivity of its heart to 2,4-D, compared with the heart of the beetle, where no gap junctions were identified.
A 48h LC(50) values of (2,4-dichlorophenoxy)acetic acid (2,4-D), a widely used agricultural herbicide, were determined on the larvae and adult Nile tilapia (Oreochromis niloticus L.). Each test was repeated three times. The data obtained were statistically evaluated by the use of the E.P.A computer program based on Finney's probit analysis method and a 48h LC(50) value for Nile tilapia (O. niloticus L.) larvae and adults were found to be 28.23mg/L and 86.90mg/L, respectively in a static bioassay test system. 95% lower and upper confidence limits for the LC(50) were 22.55-32.98 and 80.67-92.80mg/L, respectively. Water temperature was 24±1°C. Behavioral changes of both tilapia life forms were examined for various herbicide concentrations.
Tetryl (N-methyl-N,2,4,6-tetranitroaniline) is a booster explosive that was used in the production of detonators and blasting caps. It is an environmental contaminant that is found in detectable levels in areas associated with its production, use, storage, and disposal. Preliminary microsomal assays showed that one major metabolite was formed under anaerobic and aerobic conditions with both NADH and NADPH as cofactors. Metabolite formation was not inhibited by carbon monoxide but did not form in the absence of cofactor or with heat-killed microsomes. The major metabolite was identified as N-methyl-2,4,6-trinitroaniline (NMPA) by IR spectroscopy, (1)H and (14)C NMR, and chemical ionization/MS. Kinetic parameters of NMPA formation in the microsomal fraction were determined using Lineweaver-Burke plots. A V(max) of 448nmoles/(minmg) of protein and K(m) of 1.25mM was determined when NAD+ was the cofactor. When NADP+ was the cofactor, a V(max) of 139nmoles/(minmg) of protein and a K(m) of 1.4mM was determined. In the microsomal fraction, inhibition studies revealed that NMPA formation was slightly inhibited (10%) by 2'-AMP (2mM) when NADP+, but not NAD+, was used as a cofactor. This suggests that NMPA formation is partially dependent on cytochrome-P450 reductase. NMPA formation was also inhibited by dicumarol (2mM) when NADP+ (14%) and NAD+ (84%) (14%) were cofactors, suggesting that NAD(P)H: quinone oxidoreductase catalyzes NMPA formation in the microsomes. A nonspecific flavoprotein inhibitor, DPI, inhibited NMPA formation (91%) using NADP+ as a cofactor, but not NAD+. Other inhibitors, miconazole (cytochrome-P450), methimazole (flavin monooxygenase), and propylthiouracil (NADH: b5 reductase), did not prevent NMPA formation in the microsomal fraction.
Exposure chronically to n-hexane produces central-peripheral axonopathy mediated by 2,5-hexanedione (HD). Studies have shown neurofilament (NF) subunit proteins are decreased substantially in cerebral cortices, optic axons, spinal cords, and sciatic nerves from HD-exposed rats. To deeply investigate the alterations in NF contents in HD neuropathy, the relative levels of NF-L, NF-M, and NF-H in rat cerebral cortex were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. HD was administrated to Wistar rats by intraperitoneal injection at dosage of 200 or 400mg/kg. Rats were sacrificed after 6 weeks of treatment, and cerebral cortices were dissected, homogenized, and used for the determination of NF subunit proteins. The results, except for supernatant NF-L and NF-M that could not be assayed, showed HD intoxication resulted in significant decreases by 32-67% (P<0.01) in NF subunits in both of the pellet and supernatant fractions of cerebral cortex homogenate. As an initial investigation to determine how such changes in NF proteins might occur, the gene expression of NF-L, NF-M, and NF-H subunit mRNA was quantified using reverse transcription-polymerase chain reaction (RT-PCR). Statistical analysis revealed that HD exposure caused a significant reduction in the expression of NF-L and NF-H gene (P<0.05 or P<0.01), while the levels of NF-M mRNA kept unaffected (P>0.05). These suggest that the observed reduction in NF gene expression might be related to diminished levels of subunit proteins, while the actual contribution might be uncertain. The functional significance of the reduced protein contents and the regulation of gene expression remain to be determined.
The cytotoxic effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) and its major metabolites were investigated in freshly isolated rat hepatocytes. Terbutol and its metabolite, especially 2,6-di-tert-butyl-4-methylphenyl carbamate (N-demethylterbutol), at a concentration of 1.0 mM resulted in a time dependent cell killing accompanied by losses of intracellular ATP, protein thiols, and glutathione (GSH) and the accumulation of oxidized GSH. Supplementation of the hepatocyte suspension with 5 mM N-acetylcysteine, a precursor of intracellular GSH, inhibited the cytotoxicity of N-demethylterbutol. In mitochondria isolated from rat liver, terbutol and its metabolites impaired respiration related to oxidative phosphorylation and the potency of their toxicity is associated with impairment of mitochondrial respiration. These results indicate that N-demethylterbutol is the most cytotoxic followed by terbutol and other metabolites, and that both the mitochondrial respiratory system and protein thiols are important targets for these compounds.
Ascorbic acid (AA) is an essential micronutrient for man, with many biological roles. It is a powerful antioxidant both directly via scavenging of reactive oxygen species and indirectly through regeneration of other antioxidant systems. Paradoxically, under certain conditions (low concentration in vitro, presence of metal ions) it can exert a pro-oxidant effect, increasing oxidative damage to lipids, DNA and protein. Herein, the effects of vitamin C both in vitro and in vivo are addressed in terms of modulation of oxidative DNA damage, gene expression and protein oxidation. The view of AA as a simple scavenger is outdated, where the arrival of new bioinformatic techniques, heralds a new dawning in our understanding of ascorbate as a potential direct or indirect modulator of gene expression.
A novel amidine analogue of chlorambucil N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furanecarboxamide hydrochloride (AB(1)), and the parent drug were compared for their effects on collagen and DNA synthesis in breast cancer MDA-MB 231 cells. IC(50) values for chlorambucil and its amidine analogue for collagen synthesis were found to be about 44 and 19μM, respectively. Increased ability of AB(1) to suppress the protein synthesis, compared to chlorambucil, was found to be related to an inhibition of prolidase activity and expression. The phenomena were probably a result of disruption of β(1)-integrin and the insulin-like growth factor-I (IGF-I) receptor mediated signaling caused by this compound. Expression of β(1)-integrin receptor, as well as focal adhesion kinase pp125(FAK) (FAK), growth-factor receptor-bound protein 2 (GRB2), son of sevenless protein 1 (Sos1) and phosphorylated mitogen activated protein kinases (MAPK), extracellular-signal-regulated kinase 1 (ERK(1)) and kinase 2 (ERK(2)) but not Src and Shc proteins was significantly decreased in cells incubated for 24h with 10μM AB(1), compared to controls. Chlorambucil in the same conditions did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. In addition, AB(1) revealed a higher antiproliferative activity than chlorambucil, accompanied by a stronger down-regulation of IGF-I receptor expression. The results were confirmed by [(3)H]thymidine incorporation assay. Incubation of the cells with 10μM AB(1) for 12 and 24h contributed to a decrease in DNA synthesis by about 33 and 46% of the control values, respectively, while in case of chlorambucil by about 23 and 29%, respectively. These data suggest that the amidine analogue of chlorambucil (AB(1)) disturbs MDA-MB 231 cell metabolism more potently than does the parent drug, chlorambucil. The mechanism of this phenomenon may be due to its stronger suppression of β(1)-integrin and IGF-I receptor signaling.
Microcystins are a family of potent hepatotoxins and liver tumor promoters produced by several genera of cyanobacteria including Microcystis, Nodularia, Anabena, Nostoc, etc. They are chemically very stable and represent a public health threat when they occur in water used for human consumption. We investigated the DNA damage effects of M. aeruginosa UTEX 2385 in mouse liver in vivo and also in mammalian cells in vitro. The DNA damage effect is compared with purified toxin microcystin-LR (MCLR) in non-hepatic cells viz. baby hamster kidney cells (BHK-21) and mouse embryo fibroblasts primary cells (MEF). Cell-free extracts of UTEX 2385 induced significant DNA fragmentation at 0.5, 1 and 2 LD(50) (32.7, 65.4 and 130.8 mg/kg, respectively) and it was also time dependent. M. aeruginosa UTEX 2385 and MCLR induced significant DNA fragmentation in BHK-21 and MEF cells at 100 and 1.0 μg/ml concentration. Electrophoretic analysis revealed necrotic DNA damage by UTEX 2385 in vivo. Both the toxins caused smear in agarose gel electrophoresis indicating the necrotic DNA damage in MEF cells, whereas, multiple DNA fragments in BHK-21 cells. The DNA damage effect of the toxin is supported by data on hepatotoxicity in vivo and cytotoxicity in vitro.
Glycoprotein of Cudrania tricuspidata Bureau (CTB glycoprotein) was isolated from CTB fruits which have been used to heal various disorders of the injury and lung as an herbal agent in Korea since long time ago. The CTB glycoprotein was identified to have a molecular weight of 75kDa and consists of carbohydrate (72.5%) and protein moiety (27.5%). To know inhibitory ability of CTB glycoprotein for inflammation mediated by reactive oxygen radicals, firstly we tested about anti-oxidative activity (DPPH, superoxide anion, and hydroxyl radicals) in cell-free system, and then evaluated changes of inflammation-related signals [intracellular reactive oxygen species (iROS), nitric oxide (NO), nuclear factor-kappa B (NF-κB), COX-2, and iNOS] in the LPS (1μg/ml)-treated RAW 264.7cells. The results in this study showed that CTB glycoprotein (100μg/ml) has a strong scavenging activity against DPPH, superoxide anion, and hydroxyl radicals without any pro-oxidant activity in vitro. In the inflammation-related signals, expression of iROS, NO, NF-κB, COX-2, and iNOS were inhibited by treatment with CTB glycoprotein (50μg/ml) in the presence of LPS (1μg/ml). Taken together, our data obtained from these experiments indicated that CTB glycoprotein suppresses expression of the inflammatory-related proteins (iNOS and COX-2) through regulation of NF-κB. Thus, we speculate that CTB glycoprotein may have therapeutic potential for inflammation-associated disorders.
MCC-555, a treatment candidate for type 2 diabetes, is a novel thiazolidinedione which has comparatively high anti-diabetic efficacy. The present study was conducted to evaluate its toxicity and toxicokinetics in beagle dogs by oral administration at doses of 0, 6.67, 20 or 40mg/kg/day for 270 days. A 30-day recovery period was included at the end of the study to evaluate the reversibility of the toxic effects. During the treatment and recovery periods, the effects of the test agent on mortality, body weight, food consumption, hematology, serum biochemistry, urinalysis, electrocardiogram (ECG), organ weights, bone marrow and histopathology were examined. There were no treatment-related mortalities. Vomiting was observed in dogs receiving 40mg/kg/day during administration, but the dogs recovered within 1h after oral administration. Significant increases in total bilirubin and alkaline phosphatase were observed in dogs receiving the 40mg/kg/day dose during the treatment period, but the levels returned toward normal during the 30-day recovery period. Mild hydropic or fatty degeneration in the liver and inflammatory cell infiltration in the hepatic lobule or portal area was also observed sporadically without a dose-dependent relationship at the end of treatment and recovery periods. The most apparent toxicity in dogs was in the digestive system. However, these toxic effects of MCC-555 were transient and reversible. The accumulation of MCC-555 after 270-day oral administration was not notable at the toxic dose of 40mg/kg/day and the no-observed-adverse-effect level (NOAEL) was 20mg/kg/day. No differences in toxicokinetics of MCC-555 were observed between male and female dogs and no significant accumulation of MCC-555 was observed in tissues after 270 days of repeated treatments. MCC-555 distribution into different organs showed a higher penetration in the liver, kidneys and testes, followed by the ovaries and uterus. Metabolites and the metabolic style of MCC-555 are to be approved.
Imidacloprid, a neonicotinoid insecticide has been in use worldwide for several years in agriculture and veterinary medicine. It is possible that residue of this compound may be recycled in the food chain and thus information regarding effects from potential exposure to it is warranted. The objective of the present study was to evaluate immunotoxic effects of imidacloprid in female BALB/c mice. Imidacloprid was administered orally daily at 10, 5, or 2.5mg/kg over 28 days. Specific parameters of humoral and cellular immune response including hemagglutinating antibody (HA) titer to sheep red blood cells (SRBC; T-dependent antigen), delayed type hypersensitivity (DTH) response to SRBC, and T-lymphocyte proliferation in response to phytohemagglutinin (PHA) were evaluated. The results showed that imidacloprid at high dose, specifically suppressed cell-mediated immune response as was evident from decreased DTH response and decreased stimulation index of T-lymphocytes to PHA. At this dose, there were also prominent histopathological alterations in spleen and liver. Histopathological analysis of footpad sections of mice revealed dose-related suppression of DTH response. Imidacloprid at low dose of 2.5mg/kg/day did not produce any significant alterations in cellular and humoral immune response and it seemed to be an appropriate dose for assessment of 'no observable adverse effects level' for immunotoxicity in BALB/c mice. The results also indicated that imidacloprid has immunosuppressive effects at doses >5mg/kg, which could potentially be attributed to direct cytotoxic effects of IMD against T cells (particularly TH cells) and that long-term exposure could be detrimental to the immune system.
Solanum nigrum L. (SNL) has been traditionally used as an herbal plant for a long time. In the present study, SNL glycoprotein showed a dose-dependent radical scavenging activity on radicals, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radical (OH), and superoxide anion (O(2)(-)). When the HT-29 cells were treated with 60μg/ml SNL glycoprotein, the cytotoxic effect was induced in a time-dependent manner. More specifically, it was more than 60% (P<0.01) after 4h, compared to the control. On the other hand, the cells treated with 100mU/ml glucose oxidase (GO) to generate the OH radical showed a cancer resistance up to 12h. Furthermore, the addition of GO to the SNL glycoprotein caused a strong cytotoxic effect, rather than a radical scavenging effect. Interestingly, when the cells were exposed to 100mU/ml GO for 4h, the DNA-binding activity of nuclear factor-kappa B (NF-κB) was increased 4.15-fold (P<0.01) compared to the control, whereas 40μg/ml SNL glycoprotein completely blocked the DNA-binding activity of OH radical-induced NF-κB by electrophoretic mobility shift assays (EMSAs). Apoptosis, according to the apoptosis assay, increased as a result of treatment with 40μg/ml SNL glycoprotein in a time-dependent manner, whereas they were weakly induced by GO in the cells. Consequently, the SNL glycoprotein may induce apoptosis through the inhibition of NF-κB activation, induced by oxidative stress in HT-29 cells.
The abilities of the gasoline additives methyl tert-butyl ether (MTBE) and tert-amyl methyl ether (TAME) to cause liver damage following oral administration, dosed alone or in combination with model hepatotoxins, were investigated in the rat. Inducibility of liver drug-metabolizing enzyme activities was also studied. Exposure to these ethers (10-20mmol/kg) for 3 days resulted in hepatomegaly (13-30%) and induction of cytochrome P450 (CYP) activity towards N-nitrosodimethylamine (NDMAD), 7-pentoxyresorufin (PROD), and 7-ethoxyresorufin (EROD). Immunoinhibition assays with monoclonal antibodies showed that the ethers were equipotent as inducers of CYP2E1 activity (2-fold increase) but not of CYP2B1, which was elevated up to 260-fold in TAME-treated rats but only by 20-fold in MTBE rats. A slight or no modifying effect was observed on the NADPH:quinone oxidoreductase (NQO1), glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UGT) activities. Alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) were elevated in blood plasma after administration of the ethers. No dramatic enhancement of liver damage could be detected by plasma enzyme analysis (ALT, AST, alkaline phosphatase, γ-glutamyltransferase) following ether administration (13.5mmol/kg) to rats pretreated with mildly hepatotoxic dosages of ethanol, pyrazole, phenobarbital, acetaminophen (paracetamol), or 13-cis-retinoic acid (13-cis-RA or isotretinoin). Plasma triglycerides increased in TAME-treated rats (1.7-fold) and in all 13-cis-RA-treated groups (2.1-2.8-fold). The findings that MTBE and TAME exhibited a clear but differential inducing effect on two ether-metabolizing CYP forms (2E1 and 2B1) with no marked effect on phase II activities may reflect the importance of these pathways in vivo. The observation that only TAME by itself induced hypertriglyceridemia while acetaminophen- and 13-cis-RA-induced hypertriglyceridemia were aggravated by both ethers, points to differences in their effects on lipid metabolism. TAME was clearly a more potent CNS depressant than MTBE. There was no marked potentiation of drug/chemical-induced acute liver damage either by MTBE or TAME.
Hair cell loss by extrusion of nearly intact cells towards the endolimphatic cavity has been reported in the vestibular epithelia of rats subchronically exposed to 3,3'-iminodipropionitrile (IDPN). To investigate whether hair cell extrusion also occurs in the auditory system, adult male Long-Evans rats were exposed to IDPN through drinking water (0.2-0.4% for 4-14 weeks), and their organ of Corti were examined by light and scanning electron microscopy. IDPN exposure caused loss and protrusion of the outer hair cells. The surface appearance of the protruding outer hair cells was similar to that of extruding vestibular hair cells. However, the former showed cell shrinkage and irregularly shaped nucleus, by contrast to the almost normal ultrastructure shown by the latter. The results indicate that outer hair cells can suffer extrusion, but the final outcome of this process (complete extrusion versus apoptosis) remains to be determined.
The action and distribution of the insecticidal dihydropyrazole RH-3421 was examined in the trout and mouse. RH-3421 antagonized the depolarizing effect of the Na(+) channel activator veratridine and inhibited K(+)-stimulated uptake of (45)Ca(++) through voltage-sensitive calcium channels in trout brain synaptosomes. RH-3421 was a weaker inhibitor of these cellular targets in fish brain compared to mammalian brain. [(14)C]RH-3421 distributed rapidly following systemic administration to trout. Trunk kidney, muscle, liver and fat are important sites of accumulation, however, accumulation of [(14)C]RH-3421 in trout brain was low and polar metabolites were only found in bile. Mice administered an equivalent dose accumulated [(14)C]RH-3421 more efficiently into brain, and overall metabolism was more extensive. In trout, the brain is unlikely to be a major site of action of dihydropyrazoles. Our data indicate that perturbation of neuronal sites outside of brain cannot be excluded as contributing to the comparatively high acute toxicity of dihydropyrazoles in fish.
This study was carried out to investigate the hepatoprotective effect of the glycoprotein isolated from Rhus verniciflua Stokes (RVS), which has traditionally been used for healing of inflammatory diseases. We evaluated the activities of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), thiobarbituric acid-reactive substances (TBARS), and antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)] activities in treatment with carbon tetrachloride (CCl(4)) in vivo. When mice were treated with CCl(4) in the absence of RVS glycoprotein, the activities of ALT, LDH, and TBARS were increased, while the antioxidant enzymes activities were decreased. However, when the mice were treated with CCl(4) in the presence of RVS glycoprotein, the activities of ALT, LDH, and TBARS were significantly reduced and SOD, CAT, and GPx activities were remarkably increased. In addition, RVS glycoprotein increased the nitric oxide (NO) production and decreased the nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) activation in CCl(4)-treated mice. Collectively, these results pointed out that RVS glycoprotein can inhibit lipid peroxidation, enhance the activities of antioxidant enzymes, increase the NO production, and decrease the NF-κB and AP-1 activations. Therefore, we speculate that RVS glycoprotein protects from liver damage through its radical scavenging ability.
This study was earned out to investigate the antioxidative and anti-apoptotic effects of glycoprotein isolated from Gardenia jasminoides Ellis fruit (GJE glycoprotein), which has been used to heal hepatic and inflammatory diseases in folk medicine. GJE glycoprotein showed a single band with a molecular weight of 27kDa on the 15% sodium dodecyl sulfate polyacrylamide gel. It consists of a carbohydrate component (57.65%) and a protein component (42.35%). GJE glycoprotein has dose-dependent scavenging activities for DPPH, lipid peroxyl, superoxide anion and hydroxyl radicals in cell-free systems. We also evaluated the protective and anti-apoptotic activities of GJE glycoprotein on the glucose/glucose oxidase (G/GO)-induced or hypoxanthine/xanthine oxidase (HX/XO)-induced cytotoxicity and apoptosis systems in NIH/3T3 cells, using 3-(4,5-diinettiylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT), DNA fragmentation and H33342/ethidium bromide staining assays, respectively. Results in this experiment showed that GJE glycoprotein has dose-dependent blocking activities against G/GO- or HX/XO-induced cytotoxicity and apoptosis. In addition, we investigated whether GJE glycoprotein blocks the activation of redox-sensitive signal mediators, protein kinase C alpha (PKCα) and nuclear factor-kappa B (NF-κB) in G/GO or HX/XO-induced apoptotic NIH/3T3 cells, using a Western blot analysis and an electrophoretic mobility shift assay (EMSA). We found that 100μg/ml GJE glycoprotein has an inhibitory effect on PKCα translocation and the DNA binding activity of (NF-κB). Here, we speculate that GJE glycoprotein is a natural antioxidant and one of the modulators of apoptotic signal pathways in NIH/3T3 cells.
The purpose of the present study was to assess do selected pesticides as well as their binary combinations act as inhibitors of P-glycoprotein (P-gp) activity of NIH 3T3 mouse fibroblasts stably transfected with human MDR1 gene (NIH 3T3/MDR1). As a result of P-gp inhibition, the increase of intracellular accumulation of a model P-gp substrate fluorescent calcein acetoxymethyl ester was measured. Pesticide and verapamil individual dose-response data were scaled and expressed as percent of maximum effect. Results showed that out of 14 pure pesticides tested, endosulfan, phosalone and propiconazole were nearly as potent as model inhibitor verapamil (EC(50)=1.5μM), while diazinon showed a lower potency of inhibiting P-gp transport activity (EC(50)=58.4μM). Concentrations of pesticides that produced the same inhibiting effect (isoboles) were combined binary. Results calculated using the isobole method revealed that diazinon caused synergistic effect in inhibiting P-gp transport activity in all combinations.
A secondary metabolite from sponges of the genus Agelas, 4,5-dibromopyrrole-2-carboxylic acid, which is well known as feeding deterrent, was investigated for effects on the cellular calcium homeostasis in PC12 cells. 4,5-Dibromopyrrole-2-carboxylic acid did not change intracellular calcium levels if applied alone without cell depolarization. During depolarization of the cellular membrane using high potassium solution, a dose dependent reduction of intracellular calcium elevation was revealed utilizing Fura II as calcium indicator. Significant reduction was seen at concentrations higher than 30μM in a series of experiments, but in single experiments a concentration of 300nM was still reversible effective. In the same concentration range, the onset of depolarization induced calcium elevations was significantly delayed by 4,5-dibromopyrrole-2-carboxylic acid. Dose dependent reduction and delay of depolarization evoked calcium elevations are probably due to a reduction of calcium entry via voltage operated calcium channels. One cellular mode of action of the feeding deterrent potential of 4,5-dibromopyrrole-2-carboxylic acid to fishes may be an interaction with the cellular calcium homeostasis of exposed cells.
Hepatic microsomes of Aroclor 1254-treated Mugil capito showed a single protein band detected in immunoblot with monoclonal antibody 1-12-3 to teleost (scup) CYP1A. The hepatic CYP1A like protein was induced with dose dependency after exposure of the fish to β-naphthoflavone (BNF) as well as to Aroclor 1254. The induced mullet hepatic CYP1A protein was confined to a distinct fraction obtained by DE-52 anion exchange chromatography, and its relative content in that fraction increased in fish that were treated with higher doses of inducer. EROD (7-ethoxyresorufin O-deethylase) activity in hepatic microsomes from mullet treated with various doses of BNF correlated significantly (r(2)=0.81502, P<0.01) with CYP1A content. Treatment of the mullet with low dose of Aroclor 1254 (25 mg/kg) induced only traces of CYP1A in liver microsomes (5.1±4.8 mg/kg). However, in mullet treated with the high dose of Aroclor 1254 (100 mg/kg) there was a dramatic induction in CYP1A content (408±275 pmol/mg) and this hemoprotein comprised about 83% of the total P-450 content of liver microsomes. The total level of P-450, although induced in the liver tissue, was not induced in heart tissue of Aroclor 1254 treated mullet. On the other hand, P-4501A was induced in treated mullet to a level that comprised almost all of the cardiac P-450 content. EROD activity in the heart tissue of induced mullet was characterized by low V(max) and high K(m) values (K(m)=2.35 mM, V(max)=39.5 pmol/min per mg) compared to the values recorded for the enzyme from the liver (K(m)=1.0 mM, V(max)=288.0 pmol/min per mg). Cardiac CYP1A with low catalytic activity and repression of CYP-types other then CYP1A in heart of CYP1A induced fish may be part of a mechanism aimed to preserve crucial levels of electron donors and molecular oxygen in cardiac muscle of fish exposed to CYP1A inducers.
Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have been detected in the various human tissues. The OH-PBDEs are suggested to be stronger endocrine-disrupting compounds than PBDEs, therefore the toxicological effects of OH-PBDEs had received lots of attention. However, there is no study about the carcinogenic effect of OH-PBDEs and their estrogen potencies on the tumorigenesis and development of cancer. In the present study, we found that 6-hydroxy-2,2',4',4'-tetrabromodiphenyl ether (6-OH-BDE-47), the most abundant OH-PBDE congeners in human serum, promoted the in vitro migration of lung cancer A549 and H358 cells by induction of epithelial to mesenchymal transition (EMT). This was confirmed by that 6-OH-BDE-47 significantly down regulated the expression of epithelial markers E-cadherin (E-Cad) and zona occludin-1 (ZO-1) while up regulated the mesenchymal markers vimentin (Vim) and N-cadherin (N-Cad). 6-OH-BDE-47 up regulated the protein while not mRNA levels of Snail, which was the key transcription factor of EMT. Silencing of Snail by use of siRNA attenuated the 6-OH-BDE-47 induced EMT. This suggested that the stabilization of Snail was essential for 6-OH-BDE-47 induced EMT. Further, the treatment of 6-OH-BDE-47 increased the phosphorylation of AKT and ERK in A549 cells. Only PI3K/AKT inhibitor (LY294002), but not ERK inhibitor (PD98059), completely blocked the 6-OH-BDE-47 induced up regulation of Snail and down regulation of E-Cad, suggesting that PI3K/AKT pathway is important for 6-OH-BDE-47-mediated Snail stabilization and EMT in A549 cells. Generally, our results revealed for the first time that 6-OH-BDE-47 promoted the EMT of lung cancer cells via AKT/Snail signals. This suggested that more attention should be paid to the effects of OH-PBDEs on tumorigenesis and development of lung cancer. Copyright © 2014 Elsevier B.V. All rights reserved.
Top-cited authors
Nico P.E. Vermeulen
  • Vrije Universiteit Amsterdam
Ron van der Oost
  • Waternet Institute for the Urban Water Cycle
Jonny Beyer
  • Norwegian Institute for Water Research
Caterina Faggio
  • Università degli Studi di Messina
Farhana Zahir
  • Aligarh Muslim University