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.

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.

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.

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.

Cytoprotective effects of modulators of oxidative xenobiotic metabolism in precocene II-induced hepatotoxicity.

This investigation was conducted to evaluate the effects of modulation of several phase I xenobiotic-metabolizing enzyme activities on the expression of precocene II-induced hepatotoxicity. Precocene II (175-200 mg/kg) was given intraperitoneally to male Sprague-Dawley rats that had been exposed previously to inducers (phenobarbital and 3-methylcholanthrene) or inhibitors (SKF 525-A and cimetidine) of oxidative xenobiotic metabolism. Hepatic damage was measured both biochemically (leakage of aspartate aminotransferase and alanine amino-transferase into the serum) and histologically. Significant protection from precocene II-induced hepatotoxicity was observed in all treated animals regardless of whether the modulator employed was an inducer or an inhibitor of microsomal oxidative enzymes. These results indicate that the level of activity of various forms of cytochrome P-450 significantly influences the severity of hepatic necrosis induced by precocene II. Furthermore, these results suggest that inducible non-P-450 factors, such as glutathione S-transferases, may be important in modulating precocene II-induced hepatotoxicity.

Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents.

The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs.

Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations.

A simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 X g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene.

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.

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.

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.

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.

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.

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.

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.

Toxicological significance of dihydrodiol metabolites.

Dihydrodiols are often found as the major organic-extractable metabolites of various olefinic or aromatic xenobiotics in many biological samples. Studies on the chemistry of dihydrodiol metabolites have provided insight into the pharmacokinetic behavior and the mode of action of the parent compound. The toxicology of dihydrodiol is more complex than what can be deduced solely on the basis of diminished bioavailability of the epoxide precursor, and the increased hydrophilicity associated with the dihydrodiol moiety. Dihydrodiols can be intrinsically toxic and may even represent metabolically activated species. Some of the dihydrodiol metabolites may still retain sufficient lipophilic character to serve again as substrates for microsomal oxygenases. Because of the tremendous chemical and biological diversity that existed among the various dihydrodiols, more mechanistic studies are needed to examine the toxicological properties of these compounds. It may be premature to conclude dihydrodiol formation as purely a detoxification route for xenobioties.