Toxicology

Published by Elsevier
Online ISSN: 0300-483X
Publications
Article
Metallothionein (MT) is a small-molecular weight, cysteine-rich protein that binds metals. The protective role of MT in Cd toxicity is well established but its ability to protect against toxicity of other metals remains unclear. In this study, wild-type and MT-I and -II null mice (MT-null mice) were used to determine whether MT is protective against the lethality of not only Cd but also Zn, Cu, Fe, Pb, Hg and As. Following daily subcutaneous administration of an increasing dose of each metal, starting with a low, non-toxic dose, we compared the cumulative median lethal dose (LD(50)) of each metal between wild-type and MT-null mice. The LD(50) of Cd for wild-type mice was 6.9-fold higher than for MT-null mice. The LD(50) of Zn was 2.4-fold higher for wild-type mice than for MT-null mice, and 1.4-fold higher for Cu and As. The LD(50) of Hg was 1.3-fold higher for wild-type mice than for MT-null mice, but this was not statistically significant. No difference in LD(50) values was observed between wild-type and MT-null mice following Pb and Fe administration. These results suggest that MT is an important protein in the cellular defense against Cd toxicity and lethality, but it provides much less protection against the lethality of the other metals.
 
Article
Parkinsons' disease (PD) is the most common neurodegenerative movement disorder that is a consequence of premature death of dopamine-containing neurons in the substantia nigra. A number of observations have led to the hypothesis that environmental factors, including pesticides, play a significant role in the development of PD. Among pesticides, most commonly herbicides (paraquat in particular) and insecticides have been considered. The aim of this study is to address the uncertainties provided by epidemiological studies on the role of pesticide exposures in the development of PD, with the help of experimental toxicological data. Animal models that reproduce all clinical and pathological features of human PD are not available. In addition, the fundamental questions relate to the extrapolation from experimental to actual human exposure, taking also into account the role of genetic factors. Available measurements or estimates of human exposure levels that are significantly lower than those used in animal experimentation provide little support for a causal correlation between pesticide exposure and development of PD in humans. A possible role of acute poisonings or episodes of excessive exposure, and/or of combined exposures especially at early age and/or in the presence of certain genetic variants can be hypothesised. Follow up of survivors of acute poisonings by pesticides would provide information useful in this respect. According to the available data, from a public health point of view, prevention of "high" exposures, even asymptomatic ones, especially in utero and during early age is a priority.
 
Article
The zidovudine derivative, 5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl) methoxy alaninyl phosphate (WHI-07), is a dual-function spermicidal and anti-HIV agent with contraceptive and microbicidal activity. In previous two subchronic toxicity studies, intravaginal application of 0.5-2.0% WHI-07, for up to 13 weeks, was shown to cause no local, systemic or reproductive toxicity. To evaluate the toxicity and carcinogenic potential of long-term exposure to WHl-07, groups of 50 female B(6)C(3)F(1) mice were given no treatment or exposed intravaginally to a gel-microemulsion formulation with and without 2.0% WHI-07, 5 days per week for 2 years. The dose of WHI-07 was equivalent to 5700 times its spermicidal EC(50) and 5.7x10(6) times its anti-HIV IC(50). The endpoints that were evaluated included survival, body weight, hematologic and clinical chemistry profiles, absolute and relative organ weights, and histopathology. No significant differences in mean body weight gain and survival were found among the groups of untreated control, placebo control, and 2% WHI-07-treated mice at the end of the 2-year study. The hematological and clinical chemistry profiles did not reveal any toxicologically significant changes that could be attributed to WHI-07 treatment. No clinically significant changes in absolute and relative organ weights were noted in the WHI-07 group. A variety of neoplastic and nonneoplastic lesions which were considered incidental, related to aging, or procedural, were observed in both the untreated and intravaginally treated groups. The proportion of animals with malignant tumors, the total number of malignant tumors, as well as the types of malignant tumors in the three groups was similar. The cumulative incidence of microscopic lesions in various organs showed that malignant lymphoma was the major cause of death in aging female B(6)C(3)F(1) mice, the incidence of which was unaffected by intravaginal treatment. We conclude that long-term intravaginal administration of WHI-07 is not associated with systemic toxicity or increased carcinogenicity in mice. WHI-07 has clinical potential as an active ingredient of a safe vaginal/rectal microbicide.
 
Article
Although the insecticide dichlorodiphenyltrichloroethane (DDT) was banned in the US in 1972, DDT and its major metabolite 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (DDE) are still persistent in the environment. DDE at high doses is antiandrogenic in fetal and adult rats and, therefore, is of concern in humans exposed environmentally. The objective of this work was to determine the dose-response relationship between DDE and its antiandrogenic effect in adult, male rats and to quantitate the concentration of DDE in tissues following oral exposures. Adult, male, Long-Evans rats (11-13 weeks) were castrated, implanted with testosterone capsules, and dosed by oral gavage with 0, 5, 12.5, 25, 50, or 100 mg DDE per kg body weight (BW) per day in corn oil for 4 days. On day 5 the rats were euthanized and liver, adrenals, ventral prostate, and seminal vesicles were weighed as a measure of response to DDE exposure. Blood, adrenals, brain, fat, kidney, lung, liver, muscle, ventral prostate, seminal vesicles, and skin were analyzed for DDE concentrations. Testosterone and dihydrotestosterone were measured in serum. There was a decrease in prostate weight that was not dose dependent; only the prostate weights in rats treated with 12.5 mg DDE per kg BW per day were reduced significantly compared to controls. The liver displayed a dose-dependent increase in weight that was significantly greater than control at DDE doses of 25, 50, and 100 mg/kg BW per day. Blood concentrations of DDE ranged from 0.32 to 11.3 ppm, while tissue concentrations ranged from 0.72 to 2620 ppm with the highest concentration in fat. Although DDE concentrations in the androgen-responsive tissues were higher than concentrations previously shown in vitro to inhibit androgen-receptor transcriptional activity, these concentrations did not appear to be antiandrogenic in vivo. The doses administered to the rats in this study are at least 105-fold greater than the daily, average of human dietary intake of DDE.
 
Article
Injury and cellular proliferation in the lung were examined following administration of 1,1-dichloroethylene (1,1-DCE) or vinylidene chloride. C57BL/6 male mice were treated orally with 200 mg/kg of 1,1-DCE prior to a single pulse of tritiated thymidine [( 3H]TdR). Necrosis and exfoliation of Clara cells of bronchiolar epithelium were evident by 1 day after chemical administration, and increased in severity by 2 days. A regenerative response was observed at 3 days after 1,1-DCE administration, and by 7 days the epithelium was substantially restored. At 30 days after 1,1-DCE, re-epithelization was achieved and areas devoid of epithelium were not observed. Changes in cellular proliferation were calculated from measurements of [3H]TdR incorporation into total pulmonary DNA. Activity of [3H]TdR was significantly inhibited at 1 day after chemical administration and thereafter increased: a peak of synthesis occurred between 3 and 5 days. At 7 days after 1,1-DCE administration, incorporation of [3H]TdR decreased to levels that were not significantly different from those of control animals. Autoradiographic examination of 0.5 micron thick plastic-embedded lung sections showed that [3H]TdR was incorporated into the DNA of bronchiolar epithelial cells, macrophages, interstitial, endothelial and Type II alveolar cells. However, the majority of the label was taken up by the nonciliated bronchiolar epithelial cells. The increased [3H]TdR incorporation into whole lung correlated with repopulation of bronchioles which was observed following injury. The results demonstrated that 1,1-DCE-induced damage to Clara cells of the bronchiolar epithelium was severe and rapid; re-epithelization was achieved in a relatively short time whereas differentiation was a prolonged process.
 
Article
Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted urea herbicide widely used on agricultural crops such as soy, cotton and sugar cane. In a previous long-term study this herbicide exerted carcinogenic activity on the urinary bladder mucosa of male Wistar rats. In general, the genotoxic and mutagenic potentials of Diuron are considered to be negative. The present study aimed to evaluate the mode of action of Diuron on the urinary bladder mucosa of male Wistar rats. Six-week old male Wistar rats were fed pelleted Nuvilab diet mixed with Diuron at 125, 500 and 2500 ppm. As a positive control, 8.3% sodium saccharin (NaS) was fed in the diet. Preceding the sacrifice of the animals at the 20th week, urinary pH was measured and the genotoxic potential of Diuron was evaluated by the comet assay. Histological urothelial lesions in the urinary bladder and in the renal pelvis mucosa, cell proliferation/apoptosis evaluations, and scanning electron microscopy (SEM) of the urinary bladder mucosa were also performed. No DNA changes were found in urothelial or peripheral blood cells, and urinary pH was comparable to controls in all Diuron groups. In the urinary bladder urothelium, the incidence of simple hyperplasia (SH) by light microscopy was significantly increased (7/10; p<0.005) in the 2500 ppm Diuron group but not at the lower doses. By SEM, three of five animals treated with 2500 ppm Diuron showed urothelial cell necrosis and hyperplasia. In the renal pelvis, the incidence of SH was significantly increased in the Diuron 500 and 2500 ppm and in the NaS 8.3% groups. Cell proliferation was significantly increased in the Diuron 2500 ppm (p<0.05) and NaS 8.3% (p<0.05) groups. The results indicate that a high dietary concentration of Diuron is associated with urothelial necrosis and continuous regenerative cell proliferation that leads to urothelial hyperplasia.
 
Article
To establish a paraquat-resistant Wistar rat strain, we carried out continuous sister-brother mating among rats that survived high-dose intraperitoneal administration of paraquat dichloride (360 mg/kg). The percentages of paraquat-resistant rats among wild rats and among the fifth-generations were 7.1% and 20.6%, respectively. After high-dose paraquat administration, the serum paraquat concentration in sensitive rats was much higher than that in paraquat-resistant rats. The cytosol fraction of liver from paraquat-resistant rats had higher paraquat- and diquat-metabolizing activities than that of liver from paraquat-sensitive rats. By contrast, microsomal fractions from livers of paraquat-resistant and paraquat-sensitive rats had no paraquat- or diquat-metabolizing activity. This paraquat/diquat-metabolizing enzyme was partially purified from paraquat-resistant rat liver cytosol using affinity chromatography for diquat. At the end of the purification procedure, rat liver diquat-metabolizing enzyme was purified 1154-fold to a final specific activity of 32.32 mol/h/mg protein, and an overall recovery of about 0.46% was obtained. This enzyme oxidized diquat to diquat-dipyridone during overnight incubation at 37 degrees C, but only metabolized traces of paraquat. The molecular mass of the enzyme was estimated as 190 kDa, and its isoelectric point of it was 4.6-4.7. Kinetic study revealed the values of K(m) and V(max) to be 35.0 micromol/l and 0.81 micromol/h/ml, respectively.
 
Article
Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a herbicide that induced urothelial tumors in the urinary bladder of Wistar rats fed 2,500ppm during a long-term study. The currently suggested non-genotoxic mode of action (MOA) of diuron encompasses in succession urothelial necrosis induced by direct cytotoxicity, regenerative cell proliferation and sustained urothelial hyperplasia that increases the likelihood of neoplasia development. This study evaluated the dose-response profile of urothelial histological and ultrastructural lesions induced by diuron. Sixty male Wistar rats were fed ad libitum diuron mixed in the diet at 0, 60, 125, 500, 1,250, or 2,500ppm for 20 weeks. The incidences of urothelial simple hyperplasia and the cell proliferation index were significantly increased in the diuron-fed 1,250 and 2,500ppm groups. By scanning electron microscopy, the incidences and severity of lesions were significantly increased in the 500 and 1,250ppm groups. The incidences of urothelial hyperplasia in the kidney pelvis were significantly increased in the 500, 1,250 and 2,500ppm groups. The present study documents the dose-response influence of diuron on the rat urothelium, with a no observed effect level (NOEL) at 125ppm; 1,250ppm was as effective as 2,500ppm at inducing urothelial lesions.
 
Article
Bile flow and biliary excretion of the inert solute [3H]inulin were monitored in unanesthetized, freely moving male rats for 4 h after oral administration of 1,1-dichloroethylene (1,1-DCE) at a dose of 200 mg/kg. Comparisons were made between 4 groups: fed-controls, fed-1,1-DCE treated, fasted-controls, and fasted-1,1-DCE treated. Biliary inulin excretion was assessed at 30-min intervals as total excretion and as bile/plasma ratio. 1,1-DCE treatment was consistently associated with at least a 2-fold increase in both parameters of inulin excretion within 2 h after toxin administration. In contrast, 1,1-DCE treatment was not associated with changes in plasma inulin values at any time or in liver/plasma inulin ratios at 4 h. Bile flow decreased in all groups: gradually by 30% in the fed and fasted controls, by 40% in the fed-1,1-DCE treated group, and markedly by 65% in the fasted-1,1-DCE treated group. Liver damage at 4 h as reflected by elevated plasma activities of liver-derived enzymes was found only in fasted-1,1-DCE treated rats. Thus the cholestatic effect of 1,1-DCE appears related to the development of liver damage whereas other aspects of the hepatic response to 1,1-DCE may enhanced biliary excretion of inulin.
 
Article
Our objective was to determine if the previously reported protective effect of hypothyroidism against 1,1-dichloroethylene hepatotoxicity was associated with a change in distribution and covalent binding. Sprague-Dawley male rats were made hypothyroid (HypoT) by surgical thyroidectomy 2 weeks prior to studies and compared to euthyroid (EuT) rats. Hypothyroidism decreased body weights and liver to body weight ratios while mitochondrial non-protein sulfhydryl groups and cytosolic alcohol dehydrogenase activities were increased by 50%. Rats received a single oral dose of 100 mg [14C]1,1-dichloroethylene (DCE)/kg in mineral oil and were killed at 2, 4, 12 or 24 h; controls received mineral oil only. More rapid liver injury, as measured by serum alanine aminotransferase activity and histology, was present at 2 and 4 h after DCE in HypoT than EuT rats, but a similar magnitude of injury was evident at 12 and 24 h. DCE decreased liver non-protein sulfhydryl groups to a comparable extent in HypoT and EuT rats. Cytosolic glutathione S-transferase and alcohol dehydrogenase activities were decreased only in HypoT rats after DCE. HypoT rats excreted approximately 30% less total [14C]DCE-derived label in urine and their livers, kidneys and lungs consistently contained slightly less covalently bound [14C]DCE-derived label. In contrast, between 1 and 4 h after DCE, greater amounts of acid-soluble and acid-precipitable [14C]DCE-derived label were recovered in red blood cells of HypoT rats. Our results indicate that hypothyroidism did not protect against oral DCE hepatotoxicity but was associated with a more rapid injury at early times. Concurrently, hypothyroidism was found to change the fate of [14C]DCE with higher amounts of 14C-label recovered at early times in red blood cells while less 14C-label was excreted in urine and bound to liver.
 
Article
DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) is responsible for many immuno-dysregulatory functions in exposed animals, but data particularly on complement system and macrophages are limited. In this study we have shown that DDT activates the complement system through the alternative pathway in the absence of any pathogen. A significant (p<0.05) increase in C3b, C3d and C3a generation, and decline in complement hemolytic activity was observed in insecticide exposed sera. The uncontrolled complement consumption reduces the lytic activity of the complement, which enhances the susceptibility to pyogenic infection if the exposure to DDT remains unabated. Further, DDT induced the significant (p<0.05) production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in macrophages and thus contributes inflammatory reactions, cytokine imbalance and immune-dysregulation. These molecular changes in macrophages lead to structural aberrations like heterochromatin condensation, loss of pseudopodia, cytoplasmic vacuolization, DNA fragmentation and hypodiploid nuclei as seen in our study, suggesting apoptosis. However, in presence lipopolysaccharide, DDT induced significant (p<0.05) suppression of TNF-alpha and NO generation, suggestive of impairment of macrophage microbiocidal effects. This study concludes that the functional and structural derangements of macrophages in association with uncontrolled and excessive complement consumption by DDT are perhaps one of the major mechanisms contributing to the immunosuppressive effects of insecticide.
 
Article
The effect of 1,1,1-trichloroethane (TRI) inhalation on operant response was evaluated in relation to the concentration of TRI in blood and brain tissue in mice during exposure. Male CD-1 mice were trained to lever-press for an evaporated milk reinforcer on a variable interval (VI 60) schedule for 2 h. Trained mice were then exposed to either 3500 or 5000 ppm TRI for 100 min, and the changes in the schedule-controlled performance were measured. Additional groups of mice were exposed under the same conditions as those used in the behavioral study and sacrificed at various times during exposure, and the blood and brain samples were collected and subsequently analyzed for TRI content by headspace gas chromatography. Uptake of TRI into blood and brain was rapid, with near steady-state levels reached after approximately 40-60 min of exposure. Inhalation of 5000 ppm, but not 3500 ppm TRI was seen to cause inhibition of operant response, starting approximately 30 min following the initiation of inhalation exposure and beginning to recover after 80 min of exposure. The threshold concentrations for the maximal behavioral inhibition were approximately 110 micrograms/g and 130 micrograms/ml in mouse brain and blood, respectively. It appears that in addition to TRI concentrations in blood and brain tissue, the time it takes to reach the apparent threshold TRI concentration was also a determinant for the onset of TRI neurobehavioral depression.
 
Article
The effects of a short-term in vivo administration of two liver tumour promoters (phenobarbital and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane on rat liver endoplasmic reticulum Ca(2+)-ATPase were investigated. The specific activity values of this membrane-bound enzyme significantly decreased (P less than 0.01) by 51% for phenobarbital-treated rats and by 48% for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane-treated rats compared with control animals. The depression of liver endoplasmic reticulum Ca(2+)-ATPase appears to be a manifestation of the toxicological effect of tumour promoters.
 
Article
Male Wistar rats were exposed to 200, 1000 or 2000 ppm of 1,1,2-trichloro-1,2,2-trifluoroethane vapor 5 days a week 6 h daily for 1 or 2 weeks. Proliferation and vacuolisation of the smooth endoplasmic reticulum (SER) of the liver was seen electron microscopically after 1 and 2 weeks in the rats exposed to 1000 and 2000 ppm. Among the hepatic drug metabolizing enzymes, NADPH cytochrome c reductase activity showed a dose-related decrease whereas the tightly membrane-bound UDPglucuronosyltransferase exhibited a dose-dependent enhancement in its measurable activity. The overall drug oxidation reaction, 7-ethoxycoumarin O-deethylase was not affected by the 1,1,2-trichloro-1,2,2,-trifluoroethane inhalation at all, either in the liver or in the kidneys. 1,1,2-Trichloro-1,2,2-trifluoroethane binds to cytochrome P-450 with the production of a type I difference spectrum, suggesting that it may act as a substrate for this enzyme. The binding affinity is increased by phenobarbital-treatment of the rats.
 
Article
Intoxication of male and female mice with a single dose (300 or 600 mg/kg) of 1,1,2,2-tetrachloroethane (TTCE) resulted in significant decreases in cytochrome P-450 (to 58-73% of the control) and NADPH-cytochrome (P-450) c-reductase (to 29-35% of the control) in hepatic microsomes. This was accompanied by an alteration of mixed function monooxygenases stemming from the marked reduction (to 20-64% of the control) of several oxidative activities to selected substrates towards different P-450 isozymes (classes IA1, IA2, IIB1, IIE1 and IIIA). As phase II markers, epoxide hydrolase (approximately 35% loss), UDP-glucuronosyl transferase (approximately 42% loss) and to a lesser extent glutathione S-transferase (approximately 17% loss) were all affected. Also, the activity of delta-aminolevulinic (ALA) synthetase was decreased (approximately 57% of the control). On the contrary, heme oxygenase activity was increased (up to 35%) at the maximal dose tested. The decrease of P-450-function may be explained in terms of an alteration in the rate of heme biosynthesis and degradation, provoking a loss of heme content (approximately 33%) as well as of the direct inactivation of both P-450 and reductase. Because of increasing evidence on the involvement of free radical intermediates in the case of toxicity of haloalkanes, electron spin resonance spectroscopy (ESR) spin-trapping in vivo techniques were used to characterize the possible free radical species involved in the observed liver damage. The results obtained with the spin-trap N-benzylidene-2-methylpropylamine N-oxide (phenyl t-butylnitrone, PBN) provide evidence for the formation and trapping of the CHCl2CHCl free radicals. The detection of conjugated diene signals by means of second-derivative spectrophotometry, have enabled us to show that in vivo lipid peroxidation may be one of the main mechanisms responsible for TTCE hepatotoxicity.
 
Article
A single 8-h exposure to trans-1,2-dichloroethylene (t-DCE) or cis-1,2-dichloroethylene (c-DCE) at 200 ppm (hygienic standard in workplaces) resulted in a significant increase in the hexobarbital sleeping time, the zoxazolamine paralysis time, and the metabolic formation of 4-aminoantipyrine from aminopyrine in adult female Wistar rats. Higher DCE concentrations caused a dose-dependent and substantial enhancement of these effects, the effects of c-DCE being stronger than that of t-DCE. In the course of enzyme-kinetic measurements in isolated rat liver microsomes, t-DCE proved to be a competitive inhibitor of the oxidative N-demethylation of aminopyrine and of the O-demethylation of p-nitroanisole. It is concluded from the results that the inhibition of hepatic drug metabolism is caused by a competitive and reversible interaction of the 2 DCE isomers with the mixed-function oxidase system, the interaction possibly operating at the type I binding site.
 
Article
N-methyliminodiacetato(1,2-diaminocyclohexane)-platinum(II) (MIDP) is a new third-generation water-soluble antitumor platinum complex. This study compares the effects of MIDP (3 injections of 25 mg/kg each on days 1, 5 and 9) on renal structure and function and the urinary excretion of gentamicin (GENT) with those of a single 6 mg/kg dose of cisplatin (DDP) in F-344 (Fischer) rats. GENT was given as a single dose of 30 mg/kg 7 days after DDP injection or the last MIDP injection. Rats given DDP and GENT had significantly different plasma urea nitrogen (BUN) levels (315 +/- 79 mg/dl) and creatinine clearance (0.40 +/- 0.24 ml/[min.kg]) than did the control group that was given only GENT (15 +/- 1 mg/dl and 5.5 +/- 0.6 ml/[min.kg]). MIDP did not affect renal function (BUN, 16 +/- 3 mg/dl; creatinine clearance, 6.1 +/- 1.0 ml/[min.kg]). Light microscopic examination of renal tissue from MIDP-treated rats did not reveal any evidence of cell degeneration or necrosis. Rats given GENT alone excreted 72 +/- 4% of the dose in 24 h and had plasma gentamicin levels of 19 +/- 2 ng/ml 24 h after injection. The group pretreated with DDP had lower urinary GENT excretion (31 +/- 10%) and higher plasma GENT levels (7491 +/- 3750 ng/ml). MIDP pretreatment had no effect on GENT excretion (72 +/- 8%) or plasma GENT levels (16 +/- 2 ng/ml). Thus, MIDP did not cause any measureable decrease in renal function or GENT excretion in our study. Since nephrotoxicity is a significant problem with DDP administration, further studies with MIDP are warranted.
 
Article
Previous investigations have demonstrated that 1,2-dichloroethane (DCE) poisoning affects dolichol (Dol) concentration in rat liver. Dol, a long-chain polyprenol, is considered an important membrane component: as dolichyl phosphate, it is rate limiting for the synthesis of glycoprotein; as free or fatty acid, it is highly concentrated in the Golgi apparatus (GA) where it can increase membrane fluidity and permeability, required glycoprotein maturation and secretion. DCE biotransformation may stimulate pro-oxidant events through hepatocellular glutathione depletion. Since the molecules of Dol are susceptible to oxidative degradation, the aim of this investigation is to verify whether vitamin E (vit. E) supplementation in rats is able to prevent Dol breakdown during acute DCE treatment. Before acute DCE administration (628 mg/kg body weight), a group of male Wistar rats were pretreated with vit. E (33 mg/kg body weight) for 3 days. High-performance liquid chromatography analysis has shown that within 5-60 min after DCE administration, the Dol concentration decreased in liver homogenate, cytosol, microsomes and GA. Particularly, 60 min after the treatment, Dol levels in the trans Golgi fraction were 71% lower than in controls. Rat pre-treatment with vit. E prevented the DCE-induced decrease in Dol concentrations of all liver fractions considered, in particular the reduction of total-Dol observed in the trans Golgi fraction 60 min after treatment was only 40%. These data suggest that hepatic metabolism of DCE is able to promote peroxidative attacks which lead to the degradation of Dol molecules. The pre-treatment of rats with vit. E results in a good, although not complete, prevention of total-Dol depletion after DCE poisoning.
 
Article
1,2-Epoxybutane, a short-chain epoxide used as a stabilizer in chlorinated hydrocarbon solvents, was administered by inhalation exposure as a vapor 6 h/day, 5 day/week, for 24 months at exposure concentrations of 0, 200 or 400 ppm to F344/N rats and 0, 50, or 100 ppm to B6C3F1 mice. Survival of all groups of rats was 50% or greater until week 98 but was reduced in exposed groups by the end of the study. Survival in male mice was comparable among groups. Survival in female mice was greater than 50% until week 86, but was then reduced in the high-exposure group of mice. Exposure-related inflammatory, degenerative, and proliferative lesions occurred in the nasal cavity of both rats and mice. Seven papillary adenomas occurred in the nasal passages of high-exposure male rats and 2 in the nasal passages of high-exposure female rats. Alveolar/bronchiolar adenoma or carcinoma (combined) occurred with increased incidence in exposed male rats relative to controls. No exposure-related neoplastic lesions were seen in mice. After inhalation exposure, 1,2-epoxybutane was carcinogenic in rodents as were other epoxides or related compounds including propylene oxide, 1,3-butadiene, and ethylene oxide. The site of carcinogenic activity was considered to be related to length of the carbon chain.
 
Article
An evaluation was made of the protective effects of ICRF-186 [(L)1,2-bis(3,5-dioxopiperazinyl-l-yl)propane], the L-enantiomer of ICRF-187 [(D)1,2-bis(3,5-dioxopiperazinyl-l-yl)propane], against the cardiotoxicity and nephrotoxicity induced in spontaneously hypertensive rats (SHR) by doxorubicin. SHR were given doxorubicin (1 mg/kg, i.v.), once a week for 12 weeks. Group 1 (n = 10) received doxorubicin alone; Groups 2, 3 and 4 (each, n = 5) received ICRF-186, 25 mg/kg (group 2), 12.5 mg/kg (group 3) or 6.25 mg/kg (group 4), i.p., 30 min before each dose of doxorubicin. Two groups of control animals (each, n = 5) received 12 weekly i.p. injections of saline or 25 mg/kg ICRF-186. ICRF-186 provided significant protection, in a dose-dependent manner, against the cardiotoxicity and nephrotoxicity of doxorubicin and attenuated the increases in cardiac immune effector cells (interstitial dendritic cells, cytotoxic T-helper lymphocytes and macrophages) associated with this cardiotoxicity. The results of the study were compared with those obtained with ICRF-187 under identical experimental conditions. Analysis of the cardiomyopathy scores, nephropathy scores and counts of the numbers of immune effector cells in the heart showed that, at a dose of 25 mg/kg, ICRF-186 is a somewhat less effective protectant than ICRF-187. At a dose of 12.5 mg/kg, both compounds induced generally similar degrees of protection. At a dose of 6.25 mg/kg, both had comparable, but only minimal, protective effects.
 
Article
3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. MCPD has been reported genotoxic in vitro, and reproductive toxicity and carcinogenicity in rats. However, no previous studies have investigated MCPD-induced alterations in the immune system. In the present study, MCPD was administered by gavage for 14 days at 0, 25, 50, and 100 mg/kg per day to female Balb/c mice. The antibody-mediated immune response to sheep red blood cells (SRBC) was assessed using the antibody-forming cell (AFC) assay, and splenic cell phenotypes were quantified by flow cytometry. Hematological and histopathological changes were assessed. Mitogen-stimulated spleen lymphocyte proliferation and natural killer (NK) cell activity were evaluated. The T-lymphocyte blastogenesis by concanavalin A (Con A) or anti-CD3 and B-lymphocyte blastogenesis by lipopolysaccharide (LPS) were not significantly changed. There were no significant changes in the hematological and histopathological findings of MCPD-treated mice. However, the significant decrease in thymus weight was observed in 100 mg dose group, even though that did not change body weight gain. The cellularities of spleen and thymus were significantly reduced in high-dose group. Exposure to high dose of MCPD decreased the AFC response to SRBC in mice. There was a significant decrease in NK cell activity of mice treated with high dose of MCPD. These results indicate that MCPD could modulate the immune function in Balb/c mice.
 
Article
Environmental substances or metabolites induce neuronal damage through oxidative stress. Environmental organic solvent metabolite, 1,2-diacetylbenzene (1,2-DAB), treated rats develop limb weakness with neuropathological damage in both the central and peripheral nervous systems. In this experiment, we examined the relevance of 1,2-DAB-induced toxicity to increased oxidative stress using human dopaminergic neuroblastoma SHSY5Y cells. 1,2-DAB (4, 16, and 32 microM) disrupted cytoskeletal integrity and caused morphological changes. 1,2-DAB significantly decreased cell viability and induced cell cycle arrest in the G(1) phase in a concentration-dependent manner. At higher concentration, it produced apoptosis. Pre-treatment of cells with the antioxidants, GSH or N-acetylcysteine (NAC), effectively blocked 1,2-DAB-mediated cytotoxicity including cell viability, and morphological changes. These results therefore suggest that oxidative stress is involved in environmental metabolite 1,2-DAB-mediated neurotoxicity and that antioxidant treatment can effectively protect the nervous system from environmental hazards.
 
Article
Trichloroethylene (TCE, CAS 79-01-6) is a widely used industrial chemical, and a common environmental pollutant. TCE is a well-known carcinogen in rodents and is classified as "probably carcinogenic to humans". Several analytical methods have been proposed for detection of TCE metabolites in biological media utilizing derivatization-free techniques; however, none of them is suitable for simultaneous detection of both oxidative metabolites and glutathione conjugates of TCE in small volume biological samples. Here, we report a new combination of methods for assessment of major TCE metabolites: dichloroacetic acid (DCA), trichloroacetic acid (TCA), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG). First, DCA and TCA were extracted with ether. Second, the remaining aqueous fraction underwent solid phase extraction for DCVC and DCVG. Then, DCA and TCA were measured by hydrophilic interaction liquid chromatography ion exchange negative electrospray ionization tandem mass spectrometry, while DCVC and DCVG were measured by reverse phase positive electrospray ionization tandem mass spectrometry. This method was applied successfully to measure all 4 TCE metabolites in as little as 50 microl of serum from mice orally exposed to TCE (2100 mg/kg, 2h). Serum concentrations (mean+/-standard deviation) of the TCE metabolites obtained with this method are comparable or equivalent to those previously reported in the literature: DCA, 0.122+/-0.014 nmol/ml (limit of detection: 0.01 nmol/ml); TCA, 256+/-30 nmol/ml (0.4 nmol/ml); DCVG, 0.037+/-0.015 nmol/ml (0.001 nmol/ml); DCVC, 0.0024+/-0.0009 nmol/ml (0.001 nmol/ml). This method opens new opportunities to increase throughput and decrease number of animals required for mechanistic studies on TCE in rodents.
 
Article
Male Wistar rats were fed a semi-purified diet (MID - minimal inducing diet) with or without addition of 50 ppm of beta-naphthoflavone (BNF) for 1 week. After 1 week the rats were dosed with 20 mg/kg of 1,2-dimethylhydrazine (DMH) subcutaneously and killed at various time intervals from the injection. Enzyme levels were determined in microsomal and cytosolic fractions prepared from the liver and the intestinal mucosa. Feeding of BNF for 1 week caused a 6.5-fold increase of 7-ethoxyresorufin (7-ERR) deethylase in the colon as compared to the controls, but did not alter glutathione (GSH) content nor glutathione-S-transferase (GSHST) activity. Hepatic cytochrome P-450 and 7-ERR deethylation were not significantly altered by feeding of BNF at this concentration, whereas GSH and GSHST were increased by a factor of 1.6 and 2, respectively. In the DMH-dosed rats, O6-methylguanine was formed to a greater extent in the BNF-treated colon than in the controls at 1, 12 and 24 h, whilst N7-methylguanine levels were essentially the same in the induced and uninduced rats. No significant difference was found in the degree of hepatic DNA alkylation at any time points. As shown by our results, the nature of the diet would appear to be able to modulate the rate of metabolic activation of DMH and its binding to DNA in the target organ.
 
Article
The degree of toxicity caused in rats by captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) administered intraperitoneally is greater than that induced by orally administered captan. With regard to its effect on the drug-metabolizing enzymes of rat liver, the activity of aniline hydroxylase and the level of cytochrome P-450 were found to decrease in the treated rats 24 h after a single oral dose (650 mg/kg). The loss was even greater in the animals receiving diethyl maleate 1 h prior to captan. Furthermore, usual increase in the activity of drug biotransformation enzymes seen after phenobarbital treatment appears to decrease in rats dosed with this funaicide. In vitro incubations of rat liver microsomes with captan resulted in a profound loss of cytochrome P-450 and the acitivty of benzphetamine N-demethylase as well as aniline hydroxylase. Although the inhibition of drug-metabolizing enzyme activity by captan was observed in microsomal incubations with or without NADPH, a detectable amount of carbonyl sulfide (COS) was found only in the incubations that contained captan plus NADPH. Carbonyl sulfide appears to arise from a captan-derived metabolite, thiophosgene (CSCl2), which decomposes to COS in aqueous solutions and in the presence of NADPH inhibits the activity of drug biotransformation enzymes.
 
Article
S-(1,2-dichlorovinyl)-3-mercaptopropionic acid (DCV-3-MPA) was equally nephrotoxic to spontaneously-respiring and mechanically-ventilated, pentobarbital-anesthetized dogs. Its nephrotoxicity was expressed as dose-dependent changes in key renal function parameters, in proximal tubular S1, S2 and S3 cellular architecture and in the ability of the kidneys to respond maximally to ethacrynic acid, an efficacious loop diuretic. The nephrotoxicity associated with DCV-3-MPA was not the result of extrarenal actions such as hypoxemia and subsequent renal tissue hypoxia because mechanical ventilation was not protective. Four lines of evidence suggested that DCV-3-MPA was taken-up by renal proximal tubular cells like a fatty acid and metabolized by the mitochondrial beta-oxidation pathway to a reactive nephrotoxic intermediate: (i) probenecid pretreatment, which reduces the renal uptake of many organic anions but fails to do so with anions of fatty acids, failed to modify the nephrotoxicity of DCV-3-MPA; (ii) the next higher and lower homologues of DCV-3-MPA (i.e., S-(1,2-dichlorovinyl)-4-mercaptobutanoic acid (DCV-4-MBA) and S-(1,2-dichlorovinyl)-mercaptoacetic acid (DCV-MAA)) cannot yield the same reactive intermediate as DCV-3-MPA upon beta-oxidation and neither was nephrotoxic; (iii) DCV-MAA was found in plasma and urine following administration of DCV-4-MBA and (iv) the renal mitochondria were reproducibly damaged by DCV-3-MPA whereas the peroxisomes, which are also capable of performing beta-oxidation of certain fatty acids, were unscathed.
 
Article
Our previous investigations demonstrated that 1,2-dichloroethane (DCE) and chronic ethanol treatment separately are able to impair glycoprotein metabolism and secretion, and reduce dolichol concentration in liver membranes. The purpose of this study was to investigate whether chronic ethanol consumption can induce potentiation of rat liver damage due to DCE haloalkane used in several chemical processes and in agriculture. Rats were given 36% of their total energy as ethanol in the Lieber-DeCarli liquid diet for 8 weeks (CH group). The pair-fed control group received an isocaloric amount of dextrine-maltose (PF group). "In vitro" experiments: the DCE (6.5 mM) treatment of isolated hepatocytes from CH rats enhanced glycoprotein retention and further reduced glycoprotein secretion and 14C-glucosamine incorporation compared to the hepatocytes from CH or from PF and DCE treated rats. "In vivo" experiments: a marked decrease of dolichol concentration in microsomes (in which dolichyl phosphate is rate-limiting for the initial glycosylation of protein) and in Golgi membranes (in which total dolichol is very important for membrane permeability, fluidity and vesicle fusion) was observed in CH rats acutely treated with 628 mg/kg bw of DCE (CH+DCE) compared with CH or PF+DCE treated rats. These data suggest that chronic ethanol consumption increases DCE liver toxicity by affecting protein glycosylation processes and impairing glycolipoprotein secretion, with a concomitant retention at the level of the Golgi apparatus.
 
Article
Rat intoxication with a single dose of 1,2-dichloroethane (DCE) (50 microliters/100 g b.w) is able to induce a significant modification of protein glycosylation in the liver endoplasmic reticulum and Golgi apparatus. HPLC analysis shows that within 5-60 min after DCE-intoxication, the levels of total dolichol, free dolichol and dolichyl phosphate strongly decreased in the microsomes and Golgi apparatus. Particularly in total microsomes, dolichyl phosphate, which is rate-limiting for the biosynthesis of the N-linked oligosaccharide chains, drops to values significantly lower than in the control group 15 min after DCE poisoning. In the Golgi apparatus, the total dolichol, essential to enhance the fluidity and permeability of these membranes, early and significantly decreases already 5 min after DCE poisoning. Moreover, in the Golgi apparatus galactosyl- and sialyltransferase activities, the main enzymatic activities of terminal protein glycosylation, are significantly reduced, as measured 15 min after DCE intoxication. These data suggest that the impairment of glycoprotein synthesis, maturation and secretion may be involved in the pathogenesis of liver injury induced by acute DCE-intoxication.
 
Article
The 3-hydroxypyridin-4-ones (HPs) are iron and aluminum chelators. Their ability to enter the brain had not previously been directly determined. To determine whether they cross the blood-brain barrier (BBB), three HPs possessing a wide range of lipophilicity were examined: 1-[ethan-1'ol]-2-methyl-HP (CP40), 1,2-dimethyl-HP (CP20, L1, deferiprone), and 1,2-dimethyl-HP (CP94, EL1NEt). Their pharmacokinetics were determined in rats to establish dosing parameters for microdialysis studies of BBB permeation. Studies were then conducted with microdialysis probes in the blood, frontal cortex, and lateral ventricle to determine the rate and extent of HP BBB permeability. All three HPs were detectable in brain dialysate samples collected 0-7 min after HP injection, demonstrating rapid entry into the brain. The extent of unbound distribution (an indicator of the mechanism of BBB permeation) was 0.9 and 1.2 for the frontal cortex and lateral ventricle for CP20, and was 1.1 and 1.6 for CP94, suggesting diffusion across the BBB. The extent of unbound distribution of CP40 was 0.2 for both the frontal cortex and lateral ventricle, suggesting the presence of a transporter moving it out of brain extracellular fluid. Introduction of cyanide into the brain did not affect the brain to blood CP40 ratio, suggesting that the transporter is not energy-dependent. Both CP94 and CP40 caused death due to respiratory failure, whereas CP20 did not. The ability of less toxic bidentate HP chelators, such as CP20, to enter the brain may enable their use in the treatment of metal-induced diseases and iron-facilitated oxidative injury involving the central nervous system.
 
Article
Objective of the present study was to test the importance of tissue repair in the final outcome of S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced nephrotoxicity using colchicine (CLC) intervention. Male Swiss Webster (SW) mice were administered a normally nonlethal dose of DCVC (30 mg/kg, i.p.) on day 0 and CLC (2 mg/kg, i.p.) at 42 and 66 h after administration of DCVC. The mice were observed for mortality and various renal injury and repair parameters were studied during a time course of 0-14 days. Administration of 30 mg DCVC/kg led to loss of renal architecture by day 1, which sustained until day 5, and regressed thereafter to reach normal architecture by day 10 resulting in 100% survival. Renal dysfunction as assessed by increases in plasma BUN and creatinine levels was concordant during this time course. Urinary volume increased significantly between days 10 and 14 with significant increases in urinary glucose concentrations on days 1-4. Calpain leakage increased from day 1 and remained so until day 5 before declining at later time points. In contrast, CLC intervention led to marked inhibition of S-phase DNA synthesis and 100% mortality by 120 h. H&E sections of kidneys revealed loss of renal architecture on day 1 which progressively worsened from day 2 to 4. Polyuria and glycosuria were evident during the first 2 and 3 days, respectively. Calpain immunohistochemistry revealed progressive leakage of calpain in the extracellular space during 2-4 days which lead to increased renal injury as evident from significant increases in calpain specific breakdown products (CSBPs) of alpha-fodrin during the same period of time. The group of mice receiving 2 mg CLC/kg alone showed a significant increase in urinary creatinine concentration on day 5. Neither the expression nor localization of aquaporin 1 was altered in any of the treatment groups. These results show that antimitotic intervention after DCVC-initiated renal injury leads to expansion and progression of that injury, which appears to be due to proteolytic destruction of neighboring cells mediated by calpain leaking out of necrosed renal tubular epithelial cells.
 
Article
Intravenous doses of 92.6 and 185.2 mumol S-(1,2-dichlorovinyl)-D-cysteine (D-DCVC)/kg were acutely nephrotoxic in pentobarbital-anesthetized dogs. During the 6-h period following administration of either dose, renal arterial blood flow decreased modestly, urinary excretion rate of protein increased, and in contrast to findings in rabbits, ultrastructural lesions developed only in S1 and S2 cells of proximal tubules. The higher dose also induced significant increases in urine flow rate and urinary excretion rate of glucose. The adverse changes noted following the low dose of D-DCVC were due to its direct renal actions and not to extrarenal actions such as major changes in blood gases, total renal blood flow or mean arterial blood pressure that could have indirectly contributed to renal damage via induction of episodes of renal ischemia or hypoxia. In addition, there was a correlation between the proximal tubular cell types injured by D-DCVC and the location of D-amino acid oxidase (DAAO) in the canine nephron. Overall, the nephrotoxicity of D-DCVC was characterized by the same renal function and ultrastructure changes as noted previously with L-DCVC, but the D-isomer was slightly less potent. Our data suggested that the similarity in the toxicity of D- and L-DCVC might be related to DAAO-catalyzed conversion of D-DCVC to the corresponding alpha-ketoacid (DCV-O-MPA) and subsequent biotransformation of the latter to the same highly reactive fragment as generated from the L-isomer.
 
Article
3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. To evaluate the immunotoxicity of MCPD, we investigated its effect on the thymic subset, delayed-type hypersensitivity, mixed-lymphocyte reaction and peritoneal macrophage activity. MCPD was administered by gavage for 14 days at 0, 25, 50, and 100 mg/kg/day to female Balb/c mice. The thymic subsets and annexin-V positive cells in thymic cells were quantified by flow cytometry. Mixed-lymphocyte reaction, delayed-type hypersensitivity and peritoneal macrophage activity were assessed. The mixed-lymphocyte reaction and delayed-type hypersensitivity were not significantly changed. However, there were significant increases in the apoptosis of mice treated with high dose of MCPD compared to the vehicle control. A significant decrease in the CD4+CD8+ thymic subset of mice treated with high dose of MCPD was observed. The activity of peritoneal macrophage was significantly reduced in high dose group. These results indicate that MCPD could modulate the immune function in Balb/c mice.
 
Article
Calf thymus DNA was reacted with 1,2-epoxybutene (BDO) in phosphate buffered saline at 37 degrees C for 12 h. DNA was ethanol precipitated and hydrolyzed with 1 M HCl to release DNA bases. Adenine (A)-BDO and guanine (Gua)-BDO adducts were analyzed in the ethanol supernatant and DNA hydrolysate using LC/ESI MS/MS. Reaction of BDO with A resulted in the formation of seven A-BDO adducts with MH+ ions at m/z 206. Two of these adducts were observed in the ethanol supernatant. In addition, two formaidopyrimidine (FAPY)-adducts were detected with MH+ ions at m/z 224 in DNA hydrolysate. These data are consistent with formation of regioisomeric N-7 or N-9 adducts which can be released by spontaneous depurination or ring opening. Four other A-BDO adducts were detected in DNA hydrolysate. Similar adducts were observed for Gua. This new methodology permits simultaneous determination of initially formed adducts as well as those arising as a consequence of depurination. The latter adducts provide the opportunity for monitoring exposure to BDO through analysis of N-7 or N-3 adducts excreted in the urine.
 
Article
Significant differences in hepatotoxic injury of 1,2-dichlorobenzene (o-DCB) have been reported (Gunawardhana, L., Sipes, I.G., 1991. Dichlorobenzene hepatotoxicity strain differences and structure activity relationships. Adv. Exp. Med. Biol. 283, 731-734; Stine, E.R., Gunawardhana, L., Sipes, I.G., 1991. The acute hepatotoxicity of the isomers of dichlorobenzene in Fischer 344 and Sprague-Dawley rats: isomer specific and strain-specific differential toxicity. Toxicol. Appl. Pharmacol. 109, 472-481; Valentovic, M.A., Ball, J. G., Anestis, D., Madan E., 1993a. Acute hepatic and renal toxicity of dichlorobenzene isomers in Fischer 344 rats. J. Appl. Toxicol. 13, 1-7; Kulkarni, S.G., Duong, H., Gomila, R., Mehendale, H.M., 1996. Strain differences in tissue repair response to 1,2-dichlorobenzene. Arch. Toxicol. 70, 714-723. Kulkarni, S.G., Warbritton, A., Bucci, T., Mehendale, H.M., 1997. Antimitotic intervention with colchicine alters the outcome of o-DCB-induced hepatotoxicity in Fischer 344 rats. Toxicology. 120, 79-88). Although, hepatotoxic injury of o-DCB is greater in Fischer 344 (F344) when compared with Sprague Dawley (S-D) rats, this interstrain difference does not transcend into any difference in lethal effects of o-DCB. Interstrain difference in compensatory tissue repair has been suggested as the underlying mechanism for the lack of strain differences in lethality (Kulkarni et al., 1996; Kulkarni et al., 1997, see these refs. above). However, the mechanism(s) for this interstrain difference in tissue repair is (are) not currently understood. The objectives of the present study were (1) to investigate if the differences in compensatory tissue repair are reflected in differential protooncogene expression in S-D versus F344 rat livers and (2) to investigate if changes in protooncogene expression could explain the decrease and delay in tissue repair response beyond a threshold of 0.6 ml o-DCB/kg. Male S-D and F344 rats (8/9 weeks old) were administered either 0.6 or 1.2 ml o-DCB/kg and changes in expression of protooncogenes c-myc (immediate early) and Ha-ras (delayed early) were examined over a time course. Findings of this study indicate that the timing and extent of c-myc and Ha-ras expression varies in the two strains following administration of o-DCB. Thus, the timing and extent of compensatory liver regeneration that ensues following o-DCB administration in S-D and F344 rats is temporally concordant with the protooncogene expression in the two strains.
 
Article
1,3-Butadiene (BD) is a carcinogen in rats and mice. Previous in vitro studies showed that mouse liver microsomes formed 1,2-epoxy-3-butene (BMO) from BD and 1,2:3,4-diepoxybutane (BDE) from BMO at much higher rates than rat or human microsomes. Blood and tissue levels of BDE were significantly lower in rats than in mice following exposure to BD. Since mice are much more susceptible to cancer induced by BD than rats, these findings suggest a key role for BDE in BD-induced carcinogenicity. The aim of this study was to characterize the glutathione (GSH) conjugation of BDE by cytosol from human liver and mouse and rat liver and lung in vitro. BDE and radiolabeled GSH were incubated with cytosol. Conjugates were identified by 13C-NMR and FAB mass spectroscopy and quantitated by HPLC. The enzyme kinetics for the conjugation of BDE with GSH suggest that the higher BDE blood concentrations in mice compared with rats following inhalation exposure to BD are not due to differences in GSH conjugation of BDE.
 
Article
Activities of several glutathione-dependent enzymes, expression of cytochrome P450 isoenzymes, and time- and concentration-dependent cytotoxicity of trichloroethylene (TRI) and S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) were evaluated in primary cultures of proximal tubular (PT) and distal tubular (DT) cells from rat kidney. These cells exhibited cytokeratin staining and maintained activities of all glutathione-dependent enzymes measured. Of the cytochrome P450 isoenzymes studied, only CYP4A expression was detected. CYP4A mRNA and protein expression were higher in primary cultures of DT cells than in PT cells and were increased in DT cells by ciprofibrate treatment. Incubation of cells for 6 h with concentrations of TRI as high as 10 mM resulted in minimal cytotoxicity, as determined by release of lactate dehydrogenase (LDH). In contrast, marked cytotoxicity resulted from incubation of PT or DT cells with DCVC. Addition to cultures of TRI (2-10 mM) for 24 or 72 h resulted in modest, but significant time- and concentration-dependent increases in LDH release. Treatment of cells with DCVC (0.1-1 mM) for 24 h caused significant increases in LDH release and alterations in cellular protein and DNA content. Finally, exposure of primary cultures to TRI or DCVC for 72 h followed by 3 h of recovery caused a slight increase in the expression of vimentin, consistent with cellular regeneration. These studies demonstrate the utility of the primary renal cell cultures for the study of CYP4A expression and mechanisms of TRI-induced cellular injury.
 
Article
The metabolism and binding of 14C-labelled 1,2-dichloroethane (DCE) in female C57BL-mice were studied. As shown by whole-body autoradiography with heated and organic solvent-extracted tissue sections of i.v. injected mice, a selective localization of non-volatile and bound DCE-metabolites occurred in the nasal olfactory mucosa and the tracheo-bronchial epithelium. Low levels of metabolites were also present in the epithelia of the upper alimentary tract, vagina and eyelid, and in the liver and kidney. A decreased mucosal and epithelial binding was observed after pretreatment with metyrapone, indicating that the binding might be due to an oxidative metabolism of DCE. The quantitated levels of in vivo binding were considerably lower in mice injected i.p. with DCE, as compared to mice given equimolar doses of 14C-labelled 1,2-dibromoethane. In vitro experiments with 1000 g supernatants from various tissues showed that the nasal mucosa has a marked ability to activate DCE into products that become irreversibly bound to the tissue. It is proposed that the nasal olfactory mucosa is a target tissue for toxicity of DCE.
 
Article
The human testicular toxicant 1,2-dibromo-3-chloropropane (DBCP) was studied for the same end-point in 4 different species of laboratory animals. Marked necrosis and atrophy of the seminiferous epithelium were observed in rats and guinea pigs 10 days after a single i.p. administration of DBCP (170-340 mumol/kg), whereas significantly less damage was observed in hamsters and mice. The testicular concentrations of DBCP measured at various time-points after the i.p. injection of DBCP indicated that factors in addition to tissue concentration were of importance for the observed species differences in sensitivity towards DBCP-induced testicular damage. Also, there did not seem to be any direct correlation between DBCP-induced in vivo testicular toxicity and in vitro GSH-dependent dehalogenation, inasmuch as the rate of bromide release from DBCP with hamster testicular cytosol was as fast as that with rat cytosol. Testicular DNA damage, as determined by alkaline elution 60 min after in vivo administration of 170 mumol/kg DBCP, was observed only in rats and guinea pigs. Thus, induction of DNA damage correlates with the relative susceptibilities of the species towards DBCP-induced testicular necrosis. To further study species differences in testicular activation of DBCP to DNA-damaging intermediate(s), cells isolated from the testes of the 4 species were incubated with DBCP. Testicular cells from rats and guinea pigs were the only preparations developing substantial DNA damage after 60 min incubation with low concentrations of DBCP (5-50 microM). The findings indicate that rats are sensitive towards DBCP-induced testicular necrosis because rat testicular cells easily activate DBCP to a DNA-damaging intermediate(s). The relative high testicular DBCP concentration as well as the ability to activate DBCP may explain the sensitivity of guinea pigs towards DBCP-induced testicular toxicity.
 
Article
Treatments known to alter P-450 activity and glutathione levels were used to elucidate the involvement of P-450 and glutathione S-transferase metabolism in 1,2-dibromo-3-chloropropane (DBCP) organ toxicity in the rat. Phenobarbital pretreatment abolished DBCP-induced renal necrosis, whereas it had only a small effect on initial renal DNA damage. The DBCP levels in plasma and tissues were markedly reduced by phenobarbital pretreatment. Perdeuterated DBCP had much higher plasma and tissue levels than protio-DBCP in phenobarbital-pretreated animals, but perdeuteration was without effect in uninduced animals. This indicates that P-450 metabolism of DBCP is of major importance only in phenobarbital-pretreated animals. In order to study the effects of decreased glutathione levels on renal distribution and toxicity, rats were pretreated with either diethyl maleate or buthionine sulfoximine. The DBCP levels in plasma and tissues showed transitory elevations after diethyl maleate and buthionine sulfoximine pretreatment compared to the control situation. Despite the fact that diethyl maleate and buthionine sulfoximine pretreatments are known to block DBCP-induced DNA damage in vitro, these pretreatments did not significantly alter DBCP-induced renal necrosis nor DNA damage. Thus, a role for glutathione conjugation in DBCP-induced in vivo renal toxicity could not be established in the present study.
 
Article
This study examined the contribution of biotransformation by the mixed function oxidase system on hepatic and renal toxicity of 1,2-dichlorobenzene (1,2-DCB). Male Fischer 344 (F344) rats (190-250 g) were pretreated with phenobarbital (PB), beta-naphthoflavone (BNF), pyridine (PYR), piperonyl butoxide (PiBx) or vehicle prior to the administration of 2 or 3 mmol/kg of 1,2-DCB. Pair-fed control animals were treated with corn oil, (1 ml/kg). Plasma alanine amino-transaminase (ALT/GPT) was increased in a dose-dependent manner by 1,2-DCB. Pretreatment with PB, BNF or PB pretreatment prior to 1,2-DCB administration increased hepatic toxicity within 24 h. Toxicity was characterized by increased ALT/GPT activity and increased liver weight. Acute administration of 1,2-DCB produced renal alterations within 24 h. Renal toxicity was characterized by altered blood urea nitrogen (BUN) concentration and decreased renal cortical slice accumulation of p-aminohippurate (PAH) 24 h after injection of 3 mmol/kg 1,2-DCB. Pretreatment with PB, BNF or PYR increased the renal toxicity of 2 and 3 mmol/kg 1,2-DCB. Conversely, pretreatment with PiBx to inhibit P450 activity slightly decreased the hepatic and renal toxicity of 1,2-DCB. These results establish that the kidney was a target organ for 1,2-DCB toxicity and that the proximal tubule was a site of damage. Additionally, these studies indicate induction of P450 isozymes increased the hepatic and renal toxicity of 1,2-DCB. Further studies are needed to examine the specific role of P450 in generation of toxicity.
 
Article
This study investigated the effect of short curcumin treatment, a natural antioxidant on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) in mice. The incidence of aberrant crypt foci (ACF) was 100%, with 54±6 per colon, 10 weeks after the first DMH injection and reached 67±12 per colon after 12 weeks. A high level of undifferentiated goblet cells and a weak apoptotic activity were shown in dysplastic ACF. The morphological alterations of colonic mucosa were associated to severe oxidative stress ratio with 43% increase in malondialdehyde vs. 36% decrease in GSH. DMH also increased inducible nitric synthase (iNOS) mRNA transcripts (250%), nitrites level (240%) and arginase activity (296%), leading to nitrosative stress and cell proliferation. Curcumin treatment, starting at week 10 post-DMH injection for 14 days, reduced the number of ACF (40%), iNOS expression (25%) and arginase activity (73%), and improved redox status by approximately 46%, compared to DMH-treated mice. Moreover, curcumin induced apoptosis of dysplastic ACF cells without restoring goblet cells differentiation. Interestingly, curcumin induced a parallel increase in TGF-β1 and HES-1 transcripts (42% and 26%, respectively). In conclusion, the protective effect of curcumin was driven by the reduction of arginase activity and nitrosative stress. The up regulation of TGF-β1 and HES-1 expression by curcumin suggests for the first time, a potential interplay between these signalling pathways in the chemoprotective mechanism of curcumin.
 
Article
Single subcutaenous injections of 1,2-dibromo-3-chloropropane (DBCP) produced dose-dependent injury to the kidney, testis, epididymis and liver of male, Fischer 344 rats. Pretreatment with the enzyme inducer phenobarbital reduced the nephrotoxic property potency of DBCP. Serum creatinine and urea nitrogen concentrations were lower, and renal proximal tubular necrosis was less severe in phenocarbital pretreated than in non-pretreated rats subsequently injected with various amounts of DCP. 3-Methylcholanthrene (3-MC) or cobaltous chloride (CoCl2) pretreatments enhanced the dose-dependent necrogenic effects of DBCP on the kidney and, in general, potentiated the DBCP-induced elevations of serum creatinine and urea nitrogen concentrations. Pre- and post-treatment with the enzyme inhibitor piperonyl butoxide had no discernable effect on the nephrotoxic potency of DBCP.
 
Article
The influence of N-acetyl-S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L-cysteine (NAc-PCBD) on cysteine conjugate beta-lyase in female rat kidney has been examined. After a single, non-nephrotoxic dose of NAc-PCBD (3 mg/kg), cytosolic beta-lyase enzyme activity was increased 1.5 to 3-fold commensurate with a corresponding increase in enzyme protein levels as assessed by both Western blot and ELISA analyses. Using a cDNA probe for beta-lyase, this induction was found to be accompanied by an increase in the cognate mRNA. In contrast, a higher, nephrotoxic dose of NAc-PCBD (10 mg/kg) decreased all the above parameters. These effects appeared to be specific to the cytosolic form of the enzyme as no changes in kidney mitochondrial beta-lyase or enzyme protein levels were observed. Repeated dosing with the lower dose level (3 mg/kg) resulted in either no change, or in some instances, a reduction in the above parameters, suggesting an accumulation of the xenobiotic and a masking of the induction phenomenon. The molecular mechanisms underlying these observations are discussed in terms of the nephrotoxicity of halogenated xenobiotics.
 
Article
The potency of gamma-1,2,3,4,5,6-hexachlorocyclohexane (lindane) as a convulsant was examined in rats by infusion into a tail vein of the unrestrained animal using a lipid emulsion as vehicle and a dose rate of 200 micrograms/min. The concentration of drug in brain rose linearly up to a convulsant level, the rate of rise increasing with relative organ mass. The decline from a convulsant level was biphasic with half times of, respectively, 30 min and 3 days. Convulsant concentrations (CC) in brain were within the range obtained with other modes of administration and exhibited normal distribution but there was a moderate influence of sex, the ovarian cycle, and age on the individual response. The minimally effective and the mean convulsant doses recorded in the survey were, respectively, 1.5 and 2.4 mg/kg and, thus, of the same order as doses reportedly convulsant in the American cockroach as well as, under certain circumstances, in man. In the neonate, the mean CC in brain was 2.5 as against 4.5 micrograms/g wet weight in the adult. An argument is put forward to support the contention that this difference might be largely accounted for by the maturational increase in the lipid content of the brain. The argument uses an approximation to the mean CC in brain water which is 70 nmol/l in both the young suckling and the adult and corresponds to concentrations in which the drug reportedly effects neuroexcitation in the cockroach nervous system or binds to the ionophore of GABA-A receptors in rat brain membranes. The findings call for convulsive states in children after dermal application of lindane to be considered in terms of individual factors favouring rapid absorption as well as in terms of individual supersensitivity of the nervous system.
 
Article
In this study we assessed the relative toxicity and potency of the chlorinated naphthalenes 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) and 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Chemicals were administered in corn oil:acetone (99:1) by gavage to female Harlan Sprague-Dawley rats at dosages of 0 (vehicle), 500, 1500, 5000, 50,000 and 500,000 ng/kg (PCN 66 and PCN 67) and 1, 3, 10, 100, and 300 ng/kg (TCDD) for 2 weeks. Histopathologic changes were observed in the thymus, liver and lung of TCDD treated animals and in the liver and thymus of PCN treated animals. Significant increases in CYP1A1 and CYP1A2 associated enzyme activity were observed in all animals exposed to TCDD, PCN 66 and PCN 67. Dose response modeling of CYP1A1, CYP1A2 and thymic atrophy gave ranges of estimated relative potencies, as compared to TCDD, of 0.0015-0.0072, for PCN 66 and 0.00029-0.00067 for PCN 67. Given that PCN 66 and PCN 67 exposure resulted in biochemical and histopathologic changes similar to that seen with TCDD, this suggests that they should be included in the WHO toxic equivalency factor (TEF) scheme, although the estimated relative potencies indicate that these hexachlorinated naphthalenes should not contribute greatly to the overall human body burden of dioxin-like activity.
 
Article
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent congener of polychlorinated dibenzo-p-dioxins. The potency of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) is only 10% of that of TCDD for typical aryl hydrocarbon receptor (AHR)-mediated effects. Acute lethality, macroscopic effects, and liver toxicity of TCDD and HxCDD were compared in male rats of the strain Han/Wistar (Kuopio; H/W), and of the lines A and B. The latter two rat lines originate from crossbreeding of H/W and Long-Evans (Turku/AB) rats. H/W and line A rats are highly resistant to acute toxicity of TCDD due to an altered AHR, while line B rats are moderately resistant due to H/W-type alleles of another, yet unidentified gene contributing to TCDD resistance ("gene B"). The rats received 200-10,000 μg/kg of either TCDD or HxCDD intragastrically and were monitored for 46 days. In all rats, the highest dose of HxCDD (10,000 μg/kg) reduced body weight more effectively than an identical dose of TCDD. Only HxCDD (10,000 μg/kg) caused gastrointestinal hemorrhage, pale (fatty) livers and death by day 15 in H/W and line A rats. In line B rats, HxCDD caused pronounced hepatic fatty degeneration, whereas TCDD induced hepatic accumulation of biliverdin and its derivatives. Both congeners induced sinusoidal distension in liver. In H/W and line A rats, the estimated LD50 values were >10,000 μg/kg and 2000-10,000 μg/kg for TCDD and HxCDD, respectively; for line B rats they were 480 μg/kg and 1000-2000 μg/kg, respectively. Thus, HxCDD was more potent than TCDD in inducing acute mortality in H/W and line A rats, contrary to what is predicted by toxic equivalency factor (TEF) values. In line B, the expected rank order of potencies prevailed. These findings suggest that in addition to the canonical AHR-mediated toxic pathways, HxCDD possesses an AHR-independent mechanism of toxicity, whose main manifestations are rapid body weight loss, mortality, fatty liver and gastrointestinal hemorrhage.
 
Article
Oxidation of MPTP by monoamine oxidase (MAO), leading to the formation of reactive metabolites, is a critical step in the expression of the nigrostriatal toxicity of this molecule. A catalytic mechanism for the 2-electron oxidation of MPTP to MPDP+ and for the further 2-electron oxidation of MPDP+ to MPP+ is proposed, involving the formation of carbon-centered radical intermediates. These radical species appear to be involved in the mechanism-based inactivation of MAO by MPTP, possibly by generating 1,4-dihydropyridine adducts with the enzyme apoprotein or its coenzyme FAD. The pathways of metabolism of MPTP in brain and peripheral tissues and the active accumulation of metabolites of MPTP in dopaminergic neurons are discussed in terms of their possible contribution to the selective cytotoxicity of the compound.
 
Article
The use of intracerebral brain dialysis in freely moving rats in neurochemical and neurotoxicological research is discussed and exemplified by studies on the neurotoxin MPTP. Intrastriatal administration of its toxic metabolite MPP+, via the dialysis tube, induced massive changes in the release of neurotransmitters and metabolites. Release enhancing effects could not be repeated by a second MPP+ perfusion and decreases in neurotransmitter or metabolite output were persistent. This indicates that MPP+ has irreversible, toxic effects on various neuortransmitter systems. The MPP+-induced release of DA has been characterized by studying the effect of pretreatment with various drugs, as well by comparison of the time courses of MPP+-induced DA release with those of amphetamine-induced DA release and of MPP+-induced lactate overflow.
 
Article
The nigrostriatal dopaminergic neurotoxicity of MPTP was prevented in mice in a dose-dependent manner by the monoamine oxidase-B (MAO-B) inhibitor deprenyl. This finding, combined with other observations, points out the important role of MAO-B in the bioactivation of MPTP. In the present study, some comparisons between MPTP and several of its structural analogs will be presented.
 
Article
Organization of the actin cytoskeleton, the cytosolic free-calcium concentrations and ATP levels were analyzed in 3T3 mouse fibroblasts treated with 0.75 or 1.5 mM MPTP. In the presence of the drug actin filaments were time- and dose-dependently disorganized, ATP level was unaffected and intracellular calcium increased within 5 s. The correlation between MPTP cytotoxicity and [Ca2+]i level emerging from these results, suggests that the primary effect of the molecule itself is on the plasma membrane's integrity for calcium ion regulation.
 
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Sidney Stohs
  • Creighton University
Debasis Bagchi
  • University of Houston
Mihir Bagchi
  • Wayne State University
John L Butenhoff
  • SaluTox LLC
Mokhtar Ibrahim Yousef
  • Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt