Effects of malathion on survival, growth, development, and equilibrium posture of bullfrog tadpoles (Rana catesbeiana).

Bullfrog (Rana catesbeiana) tadpoles were exposed to malathion in water in a 28-d static renewal test. The effects of malathion on survival, growth, development, and loss of equilibrium posture were determined. Survival was significantly decreased at malathion concentrations of 2,500 micrograms/L and higher. Development of tadpoles was delayed significantly by malathion exposure as indicated by a dose-related decrease in developmental stage over time. Development of tadpoles in the 1,000-microgram/L and higher treatment groups was significantly delayed from that observed in the control. The effects of malathion on developmental stage suggest that malathion may decrease thyroid function in tadpoles, as it does in other species. Maintenance of equilibrium posture following agitation of the test containers was significantly impaired in tadpoles in all the malathion treatment groups (500 to 3,000 micrograms/L) relative to the control. Maintenance of equilibrium posture was thus the most sensitive end point measured in this study. Loss of equilibrium posture could increase predation losses and decrease feeding in populations of bullfrog tadpoles in the field. However, concentrations of malathion that produced adverse effects in our study are higher than published, measured concentrations found in wetlands or streams.

Enhanced regional expression of glutathione S-transferase P1-1 with colocalized AP-1 and CYP 1A2 induction in chlorobenzene-induced porphyria.

A distinct, nonfocal expression pattern was observed for glutathione S-transferase P1-1 (rGSTP1-1) in rats exposed to either hexachloro-(HCB) or pentachlorobenzene (PeCB). The nonfocal expression was localized to the centrilobular region with the most intense staining nearest the central vein. A Western blot analysis revealed a 5- and 15-fold induction of rGSTP1-1 with PeCB and HCB treatment on an equal molar basis, respectively. Evaluation of porphyrin fluorescence also revealed a centrilobular accumulation with average porphyrin measurements of 0.319, 0.590, and 0.206 micrograms/g tissue for PeCB, HCB, and corn-oil controls, respectively. Due to the role of Activator Protein-1 (AP-1) in rGSTP1-1 expression and CYP 1A2 in the pathogenesis of porphyria cutanea tarda, immunohistochemical localization of c-jun, c-fos, and CYP 1A2 was also performed. Increased expression and colocalization within the liver lobule was observed for c-jun, c-fos, CYP 1A2, rGSTP1-1, and areas of porphyrin accumulation. These observations are consistent with studies that have associated the induction of GST-P with jun- and fos-related gene products.

Physiologically based pharmacodynamic modeling of an interaction threshold between trichloroethylene and 1,1-dichloroethylene in Fischer 344 rats.

Physiologically based pharmacokinetic modeling (PBPK) and gas uptake experiments have been used by researchers to demonstrate the competitive inhibition mechanism between trichloroethylene (TCE) and 1,1-dichloroethylene (DCE). Expanding on their work, we showed that this pharmacokinetic interaction was absent at levels of 100 ppm or less of either chemical in gas uptake systems. In this study, we further illustrate the presence of such an interaction threshold at the pharmacodynamic level by examining the interaction effect of either chemical on the other's ability to bind and deplete hepatic glutathione (GSH) in Fischer 344 rats. However, at this end point, the pharmacodynamic interaction is complicated by the ability of the liver to resynthesize GSH in response to its depletion. To quantitatively resolve the interaction effects on GSH content from the resynthesis effects, physiologically based pharmacodynamic (PBPD) modeling is applied. Initially, the PBPD model description of hepatic GSH kinetics was calibrated against previously published data and by gas uptake experiments conducted in our laboratory. Then, the model was used to determine the duration of the gas uptake exposure experiments by identifying the critical time point at which hepatic GSH is at a minimum in response to both chemicals. Subsequently, gas uptake experiments were designed following the PBPK/PD model predictions. In these model-directed experiments, DCE was the only chemical capable of significantly depleting hepatic GSH. The application of TCE to the rats at concentrations higher than 100 ppm obstructed the ability of DCE to deplete hepatic GSH. Since the metabolites of DCE bind to hepatic GSH, this obstruction signaled the presence of metabolic inhibition by TCE. However, TCE, at concentrations less than 100 ppm, was not effective in inhibiting DCE from significantly depleting hepatic GSH. The same observations were made when the ability of DCE to cause hepatic injury, as measured by aspartate aminotransferase serum activity, was assessed. Both conclusions validated the previous findings of the presence of the interaction threshold at the pharmacokinetic level.

Human variability in hepatic glutathione S-transferase-mediated conjugation of aflatoxin B1-epoxide and other substrates.

Hepatic cytosolic fractions prepared from 14 human donors were analysed for glutathione S-transferase (GST) activity towards synthetic aflatoxin B1-8,9-epoxide (AFBO). In addition, GST-AFBO activity of pooled human liver cytosols was compared with rat, hamster, and mouse liver cytosol GST-AFBO activities. Consistent with previous studies, human liver cytosolic GSTs exhibited little activity towards AFBO. Hepatic GST-AFBO activities of rat, hamster, and mouse were 48-, 56-, and 312-fold greater, respectively, than observed for human liver using synthetic AFBO, and 70-, 465-, and 3545-fold greater, respectively, than observed for human liver using microsomally-generated AFBO. Furthermore, there was a 58-fold variation in hepatic GST-AFBO activities among the 14 human samples using synthetic AFBO as a substrate. Large interindividual variations were also observed with respect to GST activities towards bromosulfophthalein (BSP, 92-fold variation) and 3,4-dichloronitrobenzene (DCNB, 36-fold variation). Lesser interindividual variations were observed with respect to human liver GST activities towards benzo(a)pyrene-4,5-oxide (BaPO, 9-fold variation), 1-chloro-2,4-dinitrobenzene (CDNB, 8.5-fold variation), cumene hydroperoxide (CHP, 5-fold variation), and p-nitrophenyl acetate (NPA, 4-fold variation). No correlation was found among GST-AFBO activities and the presence of GST mu as determined by enzyme-linked immunosorbent assay (ELISA) or GST-trans-stilbene oxide (TSO) catalytic activity. Our observations support those of previous studies indicating that human liver cytosolic GSTs are relatively ineffective at conjugating AFBO. Furthermore, our data indicate that humans exhibit large inter-individual differences with respect to hepatic cytosolic GST conjugation of AFBO and certain other GST substrates.

Canine exposure to herbicide-treated lawns and urinary excretion of 2,4-dichlorophenoxyacetic acid.

A recent study by Hayes et al. (J. Natl. Cancer. Inst., 83: 1226-1231, 1991) found an increased risk of malignant lymphoma associated with exposure to 2,4-dichlorophenoxyacetic acid (2,4-D) in pet dogs. We conducted a study to determine the extent to which dogs absorb and excrete 2,4-D in urine after contact with treated lawns under natural conditions. Among 44 dogs potentially exposed to 2,4-D-treated lawns an average of 10.9 days after application, 2,4-D concentrations greater than or equal to 10.0 micrograms/l were found in 33 dogs (75%) and concentrations of > or = 50 micrograms/l were found in 17 (39%). Among 15 dogs with no known exposure to a 2,4-D-treated lawn in the previous 42 days, 4 (27%) had evidence of 2,4-D in urine, 1 at a concentration of > or = 50 micrograms/l. The odds ratio for the association between exposure to a 2,4-D-treated lawn and the detection of > or = 50 micrograms/l 2,4-D in urine was 8.8 (95% confidence interval, 1.4-56.2). Dogs exposed to lawns treated within 7 days before urine collection were more than 50 times as likely to have 2,4-D at concentrations > or = 50 micrograms/l than dogs with exposure to a lawn treated more than 1 week previously (odds ratio = 56.0; 95% confidence interval, 10.0-312.2). The highest mean concentration of 2,4-D in urine (21.3 mg/l) was found in dogs sampled within 2 days after application of the herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse strain differences in glutathione S-transferase activity and aflatoxin B1 biotransformation.

Previous studies have suggested that mice are resistant to the carcinogenic effects of aflatoxin B1 (AFB1) and that this resistance is largely the result of expression of an isoenzyme of glutathione S-transferase (GST) with high activity toward AFB1-8,9-epoxide. Significant interstrain differences in cytosolic GST activities toward a variety of substrates have been reported in mice. If such differences exist for the conjugation of AFB1-8,9-epoxide, then there may be significant mouse strain differences in susceptibility to AFB1-induced hepatocarcinogenicity. The hepatic microsomal and cytosolic biotransformation of AFB1 was studied in 8 different strains of mice fed a purified diet. GST-mediated conjugation of AFB1-8,9-epoxide with glutathione and GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA) and cumene hydroperoxide (CHP) were determined with cytosolic fractions from 8-10 pooled livers. Specific activities of cytochrome-P-450-mediated oxidation of AFB1 to aflatoxin Q1 (AFQ1), aflatoxin M1 (AFM1), and aflatoxin P1 (AFP1), as well as the reactive intermediate AFB1-8,9-epoxide, were determined with hepatic microsomal fractions from each mouse strain. No striking differences in specific activity between mouse strains were observed for any of the P-450- or GST-mediated enzymatic pathways measured, although some statistically significant differences were found. GST specific activities toward AFB1-8,9-epoxide, CDNB, DCNB, ECA and CHP ranged from 1.5-2.1, 2,830-5,370, 81-144, 38-69 and 32-73 nmol/mg protein/min, respectively. The rate of formation of AFB1-8,9-epoxide ranged from 208 to 465 pmol/mg protein/min. The specific activities of AFQ1,AFM1, and AFP1 formation by microsomes ranged from 36-70, 161-326, and 252-426 pmol/mg protein/min, respectively. Mice fed a standard rodent chow diet showed evidence of microsomal and cytosolic enzyme induction when compared to mice fed a purified diet. The lack of substantial differences in enzyme specific activities between mouse strains suggests that interstrain variations in the hepatocarcinogenic effects of AFB1 in mice should not be large.

Bioactivation of aflatoxin B1 by human liver microsomes: role of cytochrome P450 IIIA enzymes.

Based on our previous observations (H. S. Ramsdell and D. L. Eaton, 1990, Cancer Res. 50, 615-620) that the proportion of aflatoxin B1 (AFB1) converted to the highly reactive AFB1-8,9-epoxide in microsomal incubations varies with substrate concentration, we have examined the hypothesis of T. Shimada and F. P. Guengerich (1989, Proc. Natl. Acad. Sci. USA 86, 462-465) that cytochrome P450 IIIA4 is principally responsible for the activation (epoxidation) of AFB1 by human liver microsomes. The initial rates of formation of AFB1-8,9-epoxide and hydroxylated AFB1 metabolites were determined in microsomes prepared from livers of organ donors (n = 14) at AFB1 concentrations of 124 and 16 microM. Microsomal oxidation of nifedipine, catalyzed primarily by P450 IIIA enzymes, was also determined by HPLC. Rates of formation of AFB1 metabolites and nifedipine oxidation were poorly correlated at either AFB1 concentration (r2 = 0.13-0.41). A somewhat better correlation between AFB1 epoxidation and nifedipine oxidation was observed at 124 microM AFB1 (r2 = 0.41) than at 16 microM AFB1 (r2 = 0.26). Treatment of pooled microsomes with troleandomycin, an apparently specific inhibitor of P450 IIIA enzymes, resulted in 35% inhibition of AFB1-8,9-epoxide formation at the high AFB1 level but had little effect at 16 microM AFB1. An antibody against rat cytochrome P450 IIIA1 significantly inhibited AFB1 epoxidation at high, but not low, AFB1 concentrations, whereas AFQ1 formation was strongly inhibited at all substrate levels examined. These results are consistent with the hypothesis that cytochrome P450 IIIA enzyme(s) can form AFB1-8,9-epoxide, but are effective at only relatively high substrate concentrations. Another P450 enzyme(s) appears to be principally responsible for AFB1-8,9-epoxide formation at the low AFB1 levels that would be typical for dietary exposures.

Mouse liver glutathione S-transferase isoenzyme activity toward aflatoxin B1-8,9-epoxide and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide.

As part of the studies of the biochemical basis for species differences in biotransformation of the carcinogen aflatoxin B1 (AFB1) and its modulation by phenolic antioxidants, we have investigated the role of mouse liver glutathione S-transferase (GST) isoenzymes in the conjugation of AFB1-8,9-epoxide. Isoenzymes of GST were purified to electrophoretic homogeneity from Swiss-Webster mouse liver cytosol by affinity chromatography and chromatofocusing. The isoenzyme fractions were characterized in terms of activity toward surrogate substrates and immunologic cross-reactivity with antisera to rat GSTs. The major isoenzymes were identified as SW 4-4, SW 3-3, and SW 1-1. The specific activity of SW 4-4 toward AFB1-8,9-epoxide was at least 50- and 150-fold greater than that of SW 3-3 and SW 1-1, respectively. Relatively high activity toward another epoxide carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, was observed with both SW 4-4 and SW 3-3. SW 1-1 had the highest activity toward 1-chloro-2,4-dinitrobenzene (CDNB) whereas SW 4-4 had relatively low CDNB activity. Following pretreatment with 0.75% butylated hydroxyanisole in the diet, the fraction of total GST contributed by SW 1-1 appeared to increase dramatically, whereas in control mice SW 3-3 constituted the predominant isoenzyme. The high GST activity of mouse liver cytosol toward AFB1-8,9-epoxide is apparently due to an isoenzyme that contributes little to the overall cytosolic CDNB activity.

Species susceptibility to aflatoxin B1 carcinogenesis: comparative kinetics of microsomal biotransformation.

The biotransformation of the potential human carcinogen aflatoxin B1 (AFB1) was studied using hepatic microsomes from the rat, mouse, monkey, and human. Initial rates of AFB1 oxidation to aflatoxins Q1, M1, and P1, as well as the reactive intermediate AFB1-8,9-epoxide, were determined using a high performance liquid chromatography assay. The rates of generation of these AFB1 metabolites were investigated at low substrate concentrations (more representative of environmental exposures) and also at high ("saturating") concentrations commonly utilized in studies in vitro. Striking differences in ratios of the metabolites were observed. At an AFB1 concentration of 124 microM, mouse and monkey microsomes had the highest rates of AFB1-8,9-epoxide formation. Primate liver microsomes formed aflatoxin Q1 in large amounts but failed to produce detectable aflatoxin P1. Determination of the rates of formation over initial AFB1 concentrations ranging from 15 to 475 microM revealed that the proportion converted to AFB1-8,9-epoxide increased at lower substrate concentrations in the case of the rat and human microsomes but not with mouse or monkey microsomes. The differences in patterns of metabolite formation with varying concentrations have implications for interspecies comparisons of carcinogenic potency of AFB1.

Modification of aflatoxin B1 biotransformation in vitro and DNA binding in vivo by dietary broccoli in rats.

Cruciferous vegetables have been shown to have anticarcinogenic effects in animals but biochemical mechanisms have not been completely elucidated. The effects of dietary broccoli on in vivo DNA binding of the hepatocarcinogen aflatoxin B1 (AFB) and in vitro formation of the putative carcinogenic intermediate, AFB-8,9-epoxide, as well as detoxification of the epoxide by conjugation with glutathione (GSH), were examined in this study. Animals were fed a purified diet, a purified diet plus 25% freeze-dried broccoli, or standard rodent chow for 3 wk. In vivo binding of AFB to hepatic DNA was determined. Biotransformation of AFB in vitro (microsomal oxidation to AFB-8,9-epoxide, as well as hydroxylated metabolites, and cytosolic GSH conjugation of AFB-8,9-epoxide generated in situ) was measured by an HPLC method that allows specific and direct determination of AFB metabolites and thus, the rates of their formation. Microsomal mixed-function oxidase and epoxide hydrolase activities and cytosolic glutathione S-transferase activities were also measured with commonly used surrogate substrates. The rate of cytosolic conjugation of AFB-8,9-epoxide was increased 2.8-fold by the broccoli diet and 2.2-fold by the chow diet. These changes were not fully reflected by increases in activity with surrogate substrates. The chow diet did not affect epoxide hydrolase activity nor glutathione S-transferase activity toward 3,4-dichloronitrobenzene or benzo[a]pyrene 4,5-oxide, whereas these activities were significantly increased by the broccoli diet. Microsomal formation of AFB-8,9-epoxide was unaffected by the dietary treatments, whereas formation of aflatoxin M1 was increased. The chow diet, but not the broccoli diet, increased the amount of aflatoxin Q1 formed from AFB. Binding of AFB to DNA in vivo was significantly lower in the broccoli group but not in the chow-fed animals. These results indicate that broccoli contains substances that cause a reduction in the binding of AFB metabolites to DNA, possibly through the induction of glutathione S-transferase(s). Broccoli and rodent chow differ in their constituents that increase levels of xenobiotic biotransformation enzymes relative to a purified diet. The results also indicate the limitations of reliance on measurements of biotransformation pathways using surrogate substrates instead of carcinogenic compounds of interest.

Microsomal metabolism of pyrrolizidine alkaloids: N-oxidation of seneciphylline and senecionine.

In vivo pretreatment of rats with phenobarbital or beta-naphthoflavone reduced the specific activity of microsomal pyrrolizidine alkaloid N-oxide formation. Heat pretreatment of microsomes under conditions intended to selectively inactivate the flavin-containing monooxygenase did not lower the rate of N-oxidation. Incubation in the presence of cytochrome P-450 inhibitors diminished the microsomal formation of N-oxide. The observations are consistent with the hypothesis that pyrrolizidine alkaloid N-oxidation

Microsomal metabolism of pyrrolizidine alkaloids from Senecio jacobaea. Isolation and quantification of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine and N-oxides by high performance liquid chromatography.

The metabolism in vitro of four pyrrolizidine alkaloids from the poisonous plant Senecio jacobaea was studied. The pyrrolizidine alkaloids jacobine, jacoline, senecionine, and seneciphylline, all macrocyclic diesters of retronecine, were incubated with rat liver microsomes. Analysis of incubation extracts by HPLC with a PRP-1 reverse phase styrene-divinylbenzene resin column revealed the presence of two major metabolites, 6,7-dihydro-7-hydroxy-l-hydroxymethyl-5H-pyrrolizine and an N-oxide derivative. Mass spectrometry was used to confirm the structure of metabolites isolated by preparative HPLC using the PRP-1 column and a C-8 reverse phase column. Quantitative HPLC analysis using the PRP-1 column allowed the comparison of the rates of formation of the dihydropyrrolizine derivative and N-oxides from the four alkaloids.

Effect of sheep rumen fermentation and methane inhibition on the toxicity of Senecio jacobaea.

Senecio jacobaea (SJ) was incubated in sheep rumen fluid-buffer mixtures to determine if metabolism and(or) detoxication of pyrrolizidine alkaloids (PA) was occurring. The nontoxic reduction metabolite, 7 beta-hydroxy-l-methylene-8 alpha-pyrrolizidine, was not detected when SJ-rumen fluid incubation extracts were subjected to high performance liquid chromatography and mass spectrographic analysis. Rats were used as assay animals in another experiment to assess the toxicity of SJ incubated in rumen fluid. Incubation treatments were: Rumen fluid (RF) from a sheep not fed SJ (RF-0); RF autoclaved before incubation (RF-0A); RF from sheep fed 50% SJ for 1 wk (RF-1); RF from a sheep fed 50% SJ for 5 wk (RF-5), and RF-5 with 5 microM iodoform in the incubation medium (RF-5I). The SJ treatments were included in rat diets at the 10% level. Dietary treatment and mean rat survival times (days) were: control, no mortality; 10% untreated SJ, 43; RF-0, 53; RF-0A, 55; RF-1, 44; RF-5, 56; RF-5I, 44, There were no significant differences in survival time. This indicates that SJ was not detoxified as a result of incubation in sheep RF in vitro, and suggests that rumen detoxification does not account for resistance of sheep to SJ. The pH and volatile fatty acid concentrations of the incubation mixtures were measured before and after incubation. Acetate/propionate and pH following incubation were respectively: RF-0A, 7.3, 4.3; RF-0, 4.2, 4.4; RF-1, 2.7, 4.5; RF-5, 2.6, 4.5; RF-5I, 2.4, 4.5. These data show that although pretreatment of the rumen fluid donor with dietary SJ and addition of iodoform to the incubation mixture favor reductive rumen metabolism, detoxification of PA from SJ does not occur during in vitro sheep rumen fermentation.

Comparison of the toxicities of Senecio jacobaea, Senecio vulgaris and Senecio glabellus in rats.

The toxicity of Senecio jacobaea, S. vulgaris and S. glabellus to rats was assessed in a feeding trial. The plants were of similar toxicity, with a plant dry matter intake of about 20% of initial body weight being a lethal dose. Gas chromatography-mass spectrometry analysis demonstrated the presence of seneciphylline, jacobine, jacozine and jacoline in S. jacobaea, senecionine and seneciphylline in S. vulgaris, and senecionine in S. glabellus. An unidentified alkaloid was also found in all three plants.

Modifications of chronic hepatotoxicity of pyrrolizidine (Senecio) alkaloids by butylated hydroxyanisole and cysteine.

The chronic hepatotoxic effects of mixed pyrrolizidine alkaloids (PAs) from the poisonous plant tansy ragwort (Senecio jacobaea) and the ability of butylated hydroxyanisole (BHA) and cysteine to alter these hepatic effects were studied in male rats. In control animals, the i.p. administration of a single dose of mixed PAs, 160 mg/kg, produced marked fibrosis, biliary hyperplasia, megalocytosis, necrosis and calcification in liver 8 weeks post injection. In contrast, consumption of 0.75% BHA diet 10 days before and 14 days after PA administration reduced the incidence and/or completely prevented the occurrence of these pathological changes. Similar treatment with 1% cysteine, however, only reduced the severity of the hepatic lesions.

Mass spectrometric studies of twenty-one metabolically important acylglycines.

The mass spectra of the methyl esters of twenty-one metabolically important N-acylglycines are reported. The spectra are discussed in terms of their fragmentations and their application to studies of metabolic diseases. In particular, the high relative abundance of the peak at m/e 30 in the spectra of many aliphatic acylglycine methyl esters was studied by using ([2,2-2H2]propionyl)glycine and high resolution mass spectrometry. This fragment was assigned to the even-electron ion [CH2 = NH2]+.

Gas chromatographic studies of twenty metabolically important acylglycines.

Methylene unit values of methyl esters of twenty acylglycines of biomedical importance on three gas chromatographic columns are described. Sixteen of them were synthesized. These methylene unit values are not only of value in diagnosis of patients of known organic acidemias, but also in detecting and characterizing those of possible "new" organic acidemias.