Covalent interaction of dehydroretronecine, a carcinogenic metabolite of the pyrrolizidine alkaloid monocrotaline, with cysteine and glutathione.

The covalent interaction of dehydroretronecine, a carcinogenic metabolite of the pyrrolizidine alkaloid monocrotaline, with cysteine and glutathione, has been investigated. Dehydroretronecine was allowed to react with cysteine and glutathione in an in vitro system of phosphate buffer solutions. The reaction products were identified structurally by chromatographic, nuclear magnetic resonance, infrared, ultraviolet, and mass-spectral analysis. These data indicate that the reaction products are the sulfhydryl-linked 7-thiocysteine-dehydroretronecine and 7-thioglutathione-dehydroretronecine. Active alkylation of sulfhydryl groups is a possible mechanism by which these alkaloids interact with cellular components.

Induction of unscheduled DNA synthesis in suspensions of rat hepatocytes by an environmental toxicant, 3,3'4,4'-tetrachloroazobenzene.

Unscheduled DNA synthesis was induced by 3,3'4,4'-tetrachloroazobenzene (TCAB)) in freshly isolated suspensions of rat hepatocytes. A dose-dependent response was demonstrated. Hepatocellular DNA was obtained after the chloroform-isoamyl alchohol-phenol extraction of the isolated nuclei. The induction of unscheduled DNA synthesis was measured by the incorporation of [3H]-thymidine in the presence of hydroxyurea as determined by the scintillation counting assay. DNA repair data obtained in this study on benzo[a]pyrene and methyl methanesulfonate are comparable to a previous report using primary cultures of hepatocytes and cesium chloride gradients. Hence, the present method offers promise as a rapid and sensitive screen for chemical carcinogens.

Induction of DNA damage and repair synthesis in freshly isolated rat hepatocytes by the proallatocidin precocene II.

The proallatocidin precocene II (6,7-dimethoxy-2,2-dimethyl-2H-benzo-[b]pyran) has previously been shown to induce centrolobular liver necrosis. Here we have examined the ability of precocene II to produce DNA damage in suspensions of freshly isolated rat hepatocytes using the alkaline elution technique with N-methyl-N'-nitro-N-nitrosoguanidine as a positive control. At concentrations (10(-4)-10(-5) M) which did not induce cytotoxicity as judged by the leakage of glutamic-oxaloacetic transaminase, precocene II was capable of producing DNA single-strand breaks. In addition, a dose-dependent DNA repair synthesis (unscheduled DNA synthesis, UDS) was detected in hepatocytes exposed to precocene II. The induction of UDS was measured by incorporation of [3H]thymidine into purified hepatic DNA via a membrane filter retention method and liquid scintillation counting. Hence, results obtained in the present study indicate the potential genotoxicity of precocene II and the utility of DNA damage and repair assays in genetic toxicology.

Toxicity of firemaster FF-1 and 2,2',4,4',5,5'-hexabromobiphenyl in cultures of C3H/10T 1/2 mammalian fibroblasts.

A procedure for purifying 2,2', 4,4',5,5'-hexabromobiphenyl (HBB) from FireMaster FF-1 in gram amounts by crystallization is presented. Following purification, the structural assignment of HBB was made by using proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopy, elemental analysis, and mass spectroscopy (MS). The growth of C3H/10T 1/2 cells treated with 5, 37, 75, and 150 microgram of HBB and FF-1 per milliliter of medium was measured at 4, 8, and 13 days following treatment. FF-1 was more toxic at 37 and 75 microgram/ml at both 4 and 8 days, but the same at 13 days. At 150 microgram/ml cell growth was completely inhibited by both compounds. Growth of cells was stimulated at 5 microgram/ml, by HBB at 4 and 8 days, and FF-1 at 8 and 12 days. HCB was compared with HBB and FF-1 for cell growth toxicity at 37 and 75 microgram/ml. At 75 microgram/ml, HCB was more toxic than HBB and FF-1 during the entire time period. At 37 microgram/ml, HCB was more toxic than HBB and FF-1 at 4 and 8 days. Cells seeded at high densities and treated with HBB for three days lost the high degree of postconfluence inhibition of cell division observed in control cultures. Cells treated with FF-1 for three days did not adhere well to the plastic growth surface. Ultrastructural features of the HBB- and FF-1-treated cells included decreased surface villi and increased lysosomes relative to the control cells.

Metabolic fate of the hepatotoxic anti-juvenile hormone precocene II in the rat.

The metabolic fate of precocene II (6,7-dimethoxy-2,2-dimethyl-2H-benzo[b]pyran), a potent anti-juvenile hormone, was examined in male Sprague-Dawley rats. When a single intraperitoneal dose of [3H]precocene II was administered, approximately 34% of the administered dose was excreted within the first 24 h. Examination of the various urinary metabolites indicated that precocene II is metabolized to a stereoisomeric cis/trans mixture of the 3,4-dihydroxy-3,4-dihydroprecocene II. In addition, a mercapturic acid derivative of precocene II was tentatively identified. Thus, it is postulated that a highly reactive 3,4-epoxide intermediate is generated in vivo by the rat during oxidative metabolism.

Excretion and distribution of two occupational toxicants, tetrachloroazobenzene and tetrachloroazoxybenzene in the rat.

The clearance profile and tissue distribution of 2 occupational toxicants, 3,3',4,4'-tetrachloroazobenzene (TCAB) and 3,3',4,4'-tetrachloroazoxybenzene (TCAOB), were examined in male Sprague-Dawley rats. TCAB was found to be cleared from the body more rapidly than TCAOB when a single dose of 14C-labeled TCAB or TCAOB was administered orally. While 66% of the administered TCAB dose was excreted via the urine and feces within the first 24 h, TCAOB-treated animals were only able to clear 37% of the administered dose by the same elimination route. The half-lives for elimination of TCAB and TCAOB were estimated to be 18 h and 34 h, respectively. Examination of the tissue distribution of the remaining radioactivity indicated that, for both compounds, the adipose tissue contained the highest level of radioactivity. The rapid elimination of TCAB and TCAOB by rats may explain in part the reduced toxicity of these 2 compounds to whole animals in comparison to the isosteric 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Thymic atrophy induced by acute exposure of 3,3',4,4'-tetrachloroazobenzene and 3,3',4,4'-tetrachloroazoxybenzene in rats.

The effects of 3,3',4,4'-tetrachloroazobenzene (TCAB) and 3,3',4,4'-tetrachloroazoxybenzene (TCAOB) on lymphoid organs in male Sprague-Dawley rats were investigated in various acute exposure studies. Significant thymic atrophy was observed in rats 10 days after the i.p. administration of either compound (on days 1 and 5) at a dose of 25 mg/kg. When 8-week-old animals were studied, the relative thymus weight was reduced to 69% and 49% of the control value by the respective treatment of TCAB and TCAOB. In 2 groups of weanling rats the same dosage of TCAOB was able to reduce the relative thymus weight to 31% and 38% of the comparable control animals. In addition, TCAOB causes a decrease in the body weight gain and a decrease in the weights of major organs in the weanling animals. This toxic response cannot be explained solely on the basis of decreased food intake since qualitatively the same results were observed in a pair-feeding experiment. The involvement of glucocorticoid hormones was rejected as the underlying mechanism since adrenalectomy was found to provide no protection towards the degenerative effect seen upon the lymphoid tissues. This investigation constitutes the first study concerning the effects of these 2 environmental and occupational toxicants on lymphoid organs.

Immunosuppression in weanling and adult Sprague-Dawley rats induced by acute exposure to 3,3',4,4'-tetrachloroazoxybenzene.

The acute immunomodulatory effects of the environmental and occupational contaminant, 3,3',4,4'-tetrachloroazoxybenzene (TCAOB), were investigated on selected immune parameters in weanling and adult Sprague-Dawley rats. Significant immunotoxic effects were found 17 days after 4 doses of 25 mg/kg TCAOB, administered i.p. The main non-immune toxic effects were decreased body, kidney, heart and testis weights, and a simultaneous increase in liver weight. The immune parameters showing significant suppression were: thymic weight, splenic plaque forming cell populations and function, pertioneal macrophage chemiluminescence, and bone marrow cellularity. Weanling animals were affected by TCAOB to a greater extent than adults on both the multiple and single dose regimens. The immunotoxic effects were found to be qualitatively similar to those of its isosteric analog, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results demonstrate that TCAOB is a very potent immunotoxic compound and may have long-term effects after a single exposure. This study is the first investigation into the effect of TCAOB on immune functions.

Alteration of precocene II-induced hepatotoxicity by modulation of hepatic glutathione levels.

Precocene II (6,7-dimethoxy-2,2-dimethyl-2H-benzo[b]pyran), an insect growth regulator that is structurally related to several naturally occurring carcinogenic and non-carcinogenic alkenylbenzenes, is genotoxic and produces hepatic centrolobular necrosis in rats. This investigation was conducted to evaluate the effects of modulation of hepatic glutathione levels on the toxicity of precocene II. Administration of a toxic dose of precocene II (175 mg/kg) to male Sprague-Dawley rats rapidly depleted hepatic GSH, produced histopathological changes in the liver, and induced increases in serum aminotransferase activity. Concurrent administration of the cysteine pro-drug L-2-oxothiazolidine-4-carboxylic acid (OTC) prevented these toxic effects of precocene II. In contrast, pretreatment of rats with DL-buthionine-SR-sulfoximine (BSO), an inhibitor of glutathione synthesis, potentiated the toxicity of an otherwise non-toxic dose of precocene II (100 mg/kg). These results indicate that glutathione is important for protection from precocene II-induced hepatotoxicity.

Metabolism studies of 3,3',4,4'-tetrachloroazobenzene. I. In vitro metabolic pathways with rat liver microsomes.

The metabolism of 3,3',4,4'-tetrachloroazobenzene (TCAB), an important environmental and occupational toxicant, by rat liver microsomes has been examined in a NADPH-generating system. The metabolic pathways were delineated by the combined use of high pressure liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The rate of [14C]TCAB metabolism using induced microsomes was found to be 381 +/- 59 pmol/min/mg microsomal protein. Approximately 13% of the radioactivity from the [14C]TCAB substrate was covalently bound to the macromolecular pellet at the end of a 2-h incubation period. In addition, three distinct TCAB metabolites were isolated from the organic extracts and subsequently identified. Experiments with carbon monoxide, 2-diethylaminoethyl 2,2-diphenylvalerate hydrochloride (SKF 525-A), and control (uninduced) microsomes indicated the participation of the cytochrome P-450-dependent monooxygenases. The biological significance of both oxidative and reductive metabolic pathways were discussed. It was suggested that the generation of a reactive arene oxide intermediate mediated by oxidative enzymes may be crucial for some of the TCAB toxic effects observed in rodent tissues.

Delayed wasting syndrome and alterations of liver gluconeogenic enzymes in rats exposed to the TCDD congener 3,3', 4,4'-tetrachloroazoxybenzene.

A delayed wasting syndrome similar to that induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was observed in male Sprague-Dawley rats exposed to 3,3', 4,4'-tetrachloroazoxybenzene (TCAOB) and 3,3',4,4'-tetrachloroazobenzene (TCAB). After a slow growth period, all treatment animals (25 mg/kg, i.p., 2 doses per week) exhibited a starvation-like syndrome characterized by reduced food intake, dramatic loss of body weight and subsequent death. Although the growth of all major organs in the treatment animals was affected, the thymus appeared severely atrophied. The growth kinetics during the earlier phase were further analyzed using serially-killed rats receiving TCAOB. In addition, TCAOB was found to markedly depress the specific activity (mumol/min/g wet liver) of glucose-6-phosphatase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate kinase in the liver. Significant changes in the levels of cytochrome P-450, glutamic-pyruvic transaminase and malic enzyme in the liver were also observed.

Precocene II nephrotoxicity in the rat.

The effects of precocene II (6,7-dimethoxy-2,2-dimethylchromene) on the kidney were examined histopathologically. Marked changes were observed in the proximal convoluted tubules, collecting tubules, and glomeruli. These changes included congestion of blood in the capillaries of the glomeruli, tubular cell degeneration, and tubular cell regeneration. In addition, blood urea nitrogen levels were elevated in precocene II-treated animals. The results indicate that precocene II is nephrotoxic in the male Sprague-Dawley rat.

Antibiosis/antixenosis in tulip tree and quaking aspen leaves against the polyphagous southern armyworm, Spodoptera eridania.

Previous studies have shown leaves of tulip tree, Liriodendron tulipifera L. (of the Magnoliaceae) and of Populus tremuloides Michx. (of the Salicaceae) to be antixenotic/antibiotic to many Lepidoptera, including one of the most polyphagous of all phytophagous insects, the southern armyworm, Spodoptera eridania Cramer (Noctuidae). We investigated the physiological responses to this phytochemical activity on neonate and late instar armyworm larvae in controlled environments with particular emphasis upon the leaf extracts containing condensed tannins and hydrolysable tannins. These tannin-containing extracts of tulip tree leaves and quaking aspen leaves were generally toxic to neonate larvae. For later instars, growth suppression was not due to digestibility-reduction, but instead to suppressed consumption rates and greatly increased metabolic (respiratory) costs as reflected in reduced biomass conversion efficiencies.

Differential responses of tiger swallowtail subspecies to secondary metabolites from tulip tree and quaking aspen.

Two subspecies of the eastern tiger swallowtail butterfly, Papilio glaucus, exhibit reciprocal inabilities to survive and grow on each other's preferred foodplant. P. g. canadensis R. & J. performs well on quaking aspen (Populus tremuloides Michx.) but not on tulip tree (Liriodendron tulipifera L.); P. g. glaucus L. performs well on tulip tree but not on quaking aspen. This study was designed to test the hypothesis that secondary metabolites in tulip tree and quaking aspen are responsible for these differential utilization abilities. We extracted and fractionated leaf constituents into different chemical classes, applied them to a mutually acceptable diet (black cherry, Prunus serotina, leaves), and bioassayed them against neonate larvae (survival) and penultimate instar larvae (survival, growth, digestibility and conversion efficiencies). For each plant species, one fraction in particular showed activity against the unadapted subspecies. One tulip tree fraction dramatically reduced survival of P. g. canadensis neonates, and reduced consumption rates, growth rates, and ECI's of fourth instar larvae. The tulip tree constituents most likely responsible for these effects are sesquiterpene lactones. One quaking aspen fraction greatly lowered survival of P. g. glaucus neonates, and decreased survival, consumption rates, growth rates and ECD's of fourth instar larvae. The compounds responsible for these results are probably simple phenols or phenolic glycosides. Surprisingly, P. g. glaucus and P. g. canadensis showed slightly poorer performance on the active tulip tree and quaking aspen fractions, respectively, indicating that even adapted insects incur a metabolic cost in the processing of their host's phytochemicals.

A rapid and simple method to quantitate chemically induced unscheduled DNA synthesis in freshly isolated rat hepatocytes facilitated by DNA retention of membrane filters.

The filter elution method used for the detection of DNA strand breaks has been modified to quantitate chemically induced DNA repair which is measured as unscheduled DNA synthesis (UDS) in suspension of freshly isolated rat hepatocytes. Our method is based on DNA purification by retention on polyvinyl chloride filters, and is capable of handling a large number of samples simultaneously. By using the present assay system, positive dose-dependent UDS data was obtained on the following carcinogens: aflatoxin B1, 2-acetylaminofluorene, 4-aminobiphenyl, 2-aminofluorene, methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, and 4-nitroquinoline-1-oxide. In contrast, non-carcinogenic biphenyl, fluorene, and sodium ascorbate did not elicit any detectable levels of UDS at all concentrations tested. Thus, UDS as measured by the present filter retention method may serve as an efficient and reliable means of screening chemical mutagens/carcinogens.

Induction of sister chromatid exchange and micronuclei in primary cultures of rat and human hepatocytes by the peroxisome proliferator, Wy-14,643.

The ability of peroxisome proliferators to induce hepatocellular carcinomas in rodents has been known since the mid 1970's, but the mechanism of tumor formation is still poorly understood. In this study, we have used primary cultures of both rat and human hepatocytes to address the question of whether the peroxisome proliferator, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643), causes genotoxic damage in hepatocytes as measured by sister chromatid exchange (SCE), micronuclei formation, and chromosomal aberrations. We have found that in rat hepatocytes the number of SCEs per chromosome increased in a dose-dependent manner from a background level of 0.7 to a maximum of 1.1 in cells exposed for 48 h to 100 microM of Wy-14,643. In contrast, no increase in SCE frequency was observed in rat hepatocytes exposed to Wy-14,643 for 3 h. A dose-dependent increase in micronuclei formation was also seen in the 48 h but not in the 3 h cultures. The maximum frequency of micronuclei formation after a 48 h exposure occurred at 20 microM Wy-14,643 and was 2.3 times that for control cells. At this concentration of Wy-14,643, the frequency of chromosomal aberrations was increased by more than 10-fold. A 48 h exposure to Wy-14,643 also significantly increased micronuclei formation in human hepatocytes, but it was less effective than in rat hepatocytes. To investigate the potential role of peroxisome proliferation in these genotoxic responses, we measured the activities of palmitoyl-CoA beta-oxidase in hepatocytes exposed for 48 h to Wy-14,643. A dose-dependent increase in palmitoyl-CoA beta-oxidase activity was observed in rat hepatocytes, but not in human hepatocytes. The SCE frequency in rat hepatocytes correlated well with the degree of peroxisome proliferation, however, the increased formation of micronuclei in both rat and human hepatocytes occurred by a mechanism that appeared to be independent of peroxisome induction. In summary, these results demonstrate that the peroxisome proliferator, Wy-14,643, causes genotoxic damage in primary cultures of both rat and human hepatocytes.

Acrylonitrile-induced sister-chromatid exchanges and DNA single-strand breaks in adult human bronchial epithelial cells.

The ability of acrylonitrile to induce cytotoxicity, sister-chromatid exchanges and DNA single-strand breaks was studied in cultured human bronchial epithelial cells. The toxic effect as determined by cloning efficiency was observed at a dose of 600 micrograms/ml but not at doses of both 150 and 300 micrograms/ml. The frequency of sister-chromatid exchange in untreated cells was 3.7 +/- 1.3 per cell. In contrast, cells treated with acrylonitrile at 150 and 300 micrograms/ml exhibited 6.6 +/- 1.3 and 10.7 +/- 1.7 sister-chromatid exchanges per metaphase, respectively. DNA single-strand breaks were induced by acrylonitrile at dose levels of 200 and 500 micrograms/ml. The genotoxic effects on human bronchial epithelial cells that were directly exposed to acrylonitrile are of interest in relation to evidence for the higher lung cancer incidence of acrylonitrile workers in epidemiological studies.

Carcinogen-macromolecular adducts as biomarkers in human cancer risk assessment.

Substantial data have been generated during the past 5 years in both experimental systems and human populations which shed light on the potential role of carcinogen-macromolecular adducts in human cancer risk assessment. The use of DNA and protein adducts is based on the fundamental concept in chemical carcinogenesis that most genotoxins are metabolized to electrophilic "ultimate" carcinogens that are capable of forming covalent adducts with cellular macromolecules. This report examines the relative usefulness and limitations of using DNA and protein adducts and related techniques for assessing human exposure to genotoxic carcinogens. Data discussed in this report clearly demonstrate that these biomarkers not only allow early detection of potential cancer hazard in humans, but they can also significantly increase the power of conventional cancer epidemiological studies in determining true causal relationships. In addition, such biomarkers can improve extrapolation of cancer risks from laboratory animals to humans or from one human population to another.