Species Differences In Carcinogenicity Research Articles

Immunohistochemistry of γ-H2AX as a method of early detection of urinary bladder carcinogenicity in mice.

Phosphorylated histone H2AX (γ-H2AX) has been demonstrated as a DNA damage marker both in vitro and in vivo. We previously reported the effects of genotoxic carcinogens in the urinary bladder of rats by immunohistochemical analysis of γ-H2AX using samples from 28-day repeated-dose tests. To evaluate the application of γ-H2AX as a biomarker of carcinogenicity in the bladder, we examined species differences in γ-H2AX formation in the urinary bladder of mice. Six-week-old male B6C3F1 mice were treated orally with 12 chemicals for 4weeks. Immunohistochemical analysis demonstrated that N-butyl-N-(4-hydroxybutyl)nitrosamine, p-cresidine and 2-acetylaminofluorene (2-AAF), classified as genotoxic bladder carcinogens, induced significant increases in γ-H2AX levels in the bladder urothelium. In contrast, genotoxic (2-nitroanisole, glycidol, N-nitrosodiethylamine and acrylamide) and non-genotoxic (dimethylarsinic acid and melamine) non-bladder carcinogens did not upregulate γ-H2AX. Importantly, 2-nitroanisole, a potent genotoxic bladder carcinogen in rats, significantly increased the proportion of γ-H2AX-positive cells in rats only, reflecting differences in carcinogenicity in the urinary bladder between rats and mice. Significant upregulation of γ-H2AX was also induced by uracil, a non-genotoxic bladder carcinogen that may be associated with cell proliferation, as demonstrated by increased Ki67 expression. 2-AAF caused γ-H2AX formation mainly in the superficial layer, together with reduced and disorganized expression of uroplakin III, unlike in rats, suggesting the mouse-specific cytotoxicity of 2-AAF in umbrella cells. These results suggest γ-H2AX is a useful biomarker reflecting species differences in carcinogenicity in the urinary bladder.

1,3-Butadiene: Biomarkers and application to risk assessment

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB) and 1,2-epoxy-3,4-butanediol (EB-diol). The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for major species differences in carcinogenicity. We have conducted extensive exposure-biomarker studies on mice, rats and humans. Using low exposures that range from current occupational levels to human exposures from tobacco smoke has provided evidence that mice are very different from humans, with mice forming ∼200 times more DEB than humans at exposures of 0.1–1.5 ppm BD. While no gender differences have been noted in mice and rats for globin adducts or N-7 guanine adducts, female rats and mice had 2–3-fold higher Hprt mutations and DNA–DNA cross-links, suggesting a gender difference in DNA repair. Numerous molecular epidemiology studies have evaluated globin adducts and Hprt mutations, SCEs and chromosomal abnormalities. None of the blinded studies have shown evidence of human genotoxicity at current occupational exposures and studies of globin adducts have shown similar or lower formation of adducts in females than males. If one calculates the EB dose-equivalents for the three species, mice clearly differ from rats and humans, being ∼44 and 174 times greater than rats and humans, respectively. These data provide a scientific basis for improved risk assessment of BD.

Exposure-response of 1,2:3,4-diepoxybutane-specific N-terminal valine adducts in mice and rats after inhalation exposure to 1,3-butadiene.

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol. The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for extensive species differences in carcinogenicity. This study presents a comprehensive exposure-response for the formation of the DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) in mice and rats. Using nano-ultra high pressure liquid chromatography-tandem-mass spectrometry allowed analysis of pyr-Val in mice and rats exposed to BD as low as 0.1 and 0.5 ppm BD, respectively, and demonstrated significant differences in the amounts and exposure-response of pyr-Val formation. Mice formed 10- to 60-fold more pyr-Val compared to rats at similar exposures. The formation of pyr-Val increased with exposures, and the formation was most efficient with regard to formation per parts per million BD at low exposures. While formation at higher exposures appeared linear in mice, in rats formation saturated at exposures > or = 200 ppm for 10 days. In rats, amounts of pyr-Val were lower after 20 days than after 10 days of exposure, suggesting that the lifespan of rat erythrocytes may be shortened following exposure to BD. This research supports the hypothesis that the lower susceptibility of rats to BD-induced carcinogenesis results from greatly reduced formation of DEB following exposure to BD.

Comparative investigation of multiple organs of mice and rats in the comet assay

Mice and/or rats are usually used to detect chemical carcinogenicity and it has been known that there are species differences in carcinogenicity. To know whether there are species difference in genotoxicity, we conducted comparative investigation of multiple organs of mice and rats in the comet assay. Since the sensitivity to xenobiotics is different for different species, we queried species difference in the genotoxic sensitivity at one equitoxic level but not at one equidose. Therefore, groups of four mice or rats were treated once intraperitoneally or orally with a chemical at highest dose without death and distinct toxic manifestation. When the death was not observed at 2000 mg/kg of a chemical, 2000 mg/kg was used for the comet study. The stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and 24 h after treatment. Among chemicals tested, benzyl acetate, chlorodibromomethane and p-chloro- o-toluidine are carcinogenic to mice but not rats, and aniline, azobenzene, o-phenylphenol Na, and d-limonene are carcinogenic to rats but not mice. Although the two species differed in genotoxicity target organs and migration values, the judgement of a positive or negative response was the same for all chemicals studied except for 2,4-dimethoxyaniline, 2,5-diaminotoluene, and p, p′-DDT when chemicals with positive responses in at least one organ are judged to be comet assay-positive. 2,4-Dimethoxyaniline and 2,5-diaminotoluene that are Ames test-positive non-carcinogens in both species were positive in one organ (urinary bladder for 2,4-dimethoxyaniline and stomach for 2,5-diaminotoluene) in rats, but negative in all mouse organs. p, p′-DDT, which is an Ames test-negative but in vitro cytogenetic test-positive hepatic carcinogen in mice and rats, was positive in multiple rat organs, but not in any mouse organ. These results suggest that species differences in genotoxicity at one equitoxic level are not consistent with species difference in carcinogenicity and that the use of both species is appropriate to indicate a carcinogenic potential in the comet assay with multiple organs, when chemicals being positive in at least one organ are judged to be comet assay-positive.

Comparison of cytogenetic effects of 3,4-epoxy-1-butene and 1,2:3,4-diepoxybutane in mouse, rat and human lymphocytes following in vitro G 0 exposures

To understand better the species differences in carcinogenicity caused by 1,3-butadiene (BD), we exposed G 0 lymphocytes (either splenic or peripheral blood) from rats, mice and humans to 3,4-epoxy-1-butene (EB) (20 to 931 μM) or 1,2:3,4-diepoxybutane (DEB) (2.5 to 320 uM), two of the suspected active metabolites of BD. Short EB exposures induced little measurable cytogenetic damage in either rat, mouse, or human G 0 lymphocytes as measured by either sister chromatid exchange (SCE) or chromosome aberration (CA) analyses. However, DEB was a potent inducer of both SCEs and CAs in G 0 splenic and peripheral blood lymphocytes. A comparison of the responses among species showed that the rat and mouse were approximately equisensitive to the cytogenetic damaging effects of DEB, but the situation for the human subjects was more complex. The presence of the GSTT1-1 gene (expressed in the erythrocytes) reduced the relative sensitivity of the lymphocytes to the SCE-inducing effects of DEB. However, additional factors also appear to influence the genotoxic response of humans to DEB. This study is the first direct comparison of the genotoxicity of EB and DEB in the cells from all three species.

Mechanism of action of the nongenotoxic peroxisome proliferators: role of the peroxisome proliferator-activator receptor alpha.

Peroxisome proliferators are a diverse group of chemicals that include several therapeutically used drugs (e.g., hypolipidemic agents), plasticizers and organic solvents used in the chemical industry, herbicides, and naturally occurring hormones. As the name implies, peroxisome proliferators cause an increase in the number and size of peroxisomes in the liver, kidney, and heart tissue of susceptible species, such as rats and mice. Long-term administration of peroxisome proliferators can cause liver cancer in these animals, a response that has been the central issue of research on peroxisome proliferators for many years. Peroxisome proliferators are representative of the class of nongenotoxic carcinogens that cause cancer through mechanisms that do not involve direct DNA damage. The fact that humans are frequently exposed to these agents makes them of particular concern to government regulatory agencies responsible for assuring human safety. Whether frequent exposure to peroxisome proliferators represents a hazard to humans is unknown; however, increased cancer risk has not been shown to be associated with long-term therapeutic administration of the hypolipidemic drugs gemfibrozil, fenofibrate, and clofibrate. To make sound judgments regarding the safety of peroxisome proliferators, the validity of extrapolating results from rodent bioassays to humans must be based on the agents' mechanism of action and species differences in biologic activity and carcinogenicity. The peroxisome proliferator-activated receptor alpha (PPARalpha), a member of the nuclear receptor superfamily, has been found to mediate the activity of peroxisome proliferators in mice. Gene-knockout mice lacking PPARalpha are refractory to peroxisome proliferation and peroxisome proliferator-induced changes in gene expression. Furthermore, PPARalpha-null mice are resistant to hepatocarcinogenesis when fed a diet containing a potent nongenotoxic carcinogen WY-14,643. Recent studies have revealed that humans have considerably lower levels of PPARalpha in liver than rodents, and this difference may, in part, explain the species differences in the carcinogenic response to peroxisome proliferators.

Disposition of butadiene monoepoxide and butadiene diepoxide in various tissues of rats and mice following a low-level inhalation exposure to 1,3-butadiene.

1,3-Butadiene (BD), a chemical used extensively in the production of styrene-butadiene rubber, is carcinogenic in Sprague-Dawley rats and B6C3F1 mice. Chronic inhalation studies revealed profound species differences in the potency and organ-site specificity of BD carcinogenesis between rats and mice. BD is a potent carcinogen in mice and a weak carcinogen in rats. Previous studies from our laboratory and others have shown marked differences between rats and mice in the metabolism of BD, which may account for species differences in carcinogenicity. The purpose of the present study was to examine the production and disposition of two mutagenic BD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), in blood and other tissues of rats and mice during and following inhalation exposures to a target concentration of 62.5 p.p.m. BD. BDO was increased above background in blood, bone marrow, heart, lung, fat, spleen and thymus tissues of mice after 2 h and 4 h exposures to BD. In rats, levels of BDO were increased in blood, fat, spleen and thymus tissues. No increases in BDO were observed in rat lungs. BDO2, the more mutagenic of the two epoxides, was increased in the blood of rats and mice at 2 and 4 h after initiation of exposure to BD. In mice, BDO2 was detected in all tissues examined immediately following the 4 h exposure. This metabolite was detected in heart, lung, fat, spleen and thymus of rats, but at levels 40- to 160-fold lower than those seen in mice. Immediately after the 4 h exposure, blood levels of BDO2 were 204 +/- 15 pmol/g for mice but were 41-fold lower for rats. In the sensitive mouse target organs, heart and lungs, levels of BDO2 exceeded BDO levels immediately after the exposure. This study shows that the levels of BD epoxides are markedly greater in the mouse BD target organs. The high concentrations of BDO2 in these organs suggest that this compound may be particularly important in BD-induced carcinogenesis. Thus, although BD is oxidatively metabolized by similar metabolic pathways in rats and mice, the substantial quantitative differences in tissue levels of mutagenic epoxides between species may be responsible for the increased sensitivity of mice to BD-induced carcinogenicity.

Species Differences in Carcinogenicity: The Role of Metabolism in Human Risk Evaluation

Species Differences in Carcinogenicity: The Role of Metabolism in Human Risk Evaluation

Species differences in carcinogenicity: The role of metabolism in human risk evaluation

The assessment of human risk from exposure to chemicals is frequently confounded by differences in response to those chemicals in laboratory animals. In many cases, the basis of the species differences is differences in metabolism and pharmacokinetics. Since metabolic and pharmacokinetic data are readily accessible in both laboratory animals and humans, risk assessments can be significantly improved if these data are incorporated into the process. Several chlorinated solvents, perchloroethylene, trichloroethylene, and methylene chloride, have been investigated, and the species differences in carcinogenicity have been shown to be the result of pharmacokinetic differences between the species. The rates of metabolism of these chemicals have been measured in rats and mice in vivo, and the species differences observed have been reproduced in vitro using tissue fractions and hepatocytes. Identical experiments have been carried out using human tissues. The results of these studies together with species-specific physiological parameters have been used in a mathematical model to predict human cancer risk over a wide range of exposures. This approach provides both an explanation for the species differences in response to these chemicals and also a more rational approach to human risk assessment.

Species differences in carcinogenicity and peroxisome proliferation due to trichloroethylene: a biochemical human hazard assessment.

Trichloroethylene (TRI) administered to mice by gavage for 10 consecutive days at doses of 50-2000 mg/kg body weight elicited dose-dependent increases (up to 700% of control values) of hepatic cyanide insensitive palmitoyl CoA oxidation (a marker of peroxisomal beta-oxidation). No effect was seen on catalase; the other peroxisomal marker examined. Similar experiments with rats demonstrated no effect of TRI on either cyanide insensitive palmitoyl CoA oxidation or catalase. A major metabolite of TRI, trichloroacetic acid (TCA) when administered by gavage for 10 consecutive days at doses of 10-200 mg/kg body weight, stimulated hepatic cyanide insensitive palmitoyl CoA oxidation in both mice (up to 500% of control) and rats (up to 650% of control). Again, no effect upon catalase activity was apparent. The kinetics of biotransformation of TRI to TCA in isolated hepatocytes was markedly species dependent. The 'intrinsic clearance' values (Vmax/Km) for TRI in mouse, rat and human hepatocytes were 3.8 X 10(-6), 1.2 X 10(-7) and 3.25 X 10(-8) L/min/10(6) cells respectively. TCA induced peroxisomal beta-oxidation in mouse and rat hepatocytes, but had no effect upon this enzyme activity in cultured human hepatocytes. It is postulated that the species difference in hepatocarcinogenicity of TRI (mouse positive; rat negative) is due to species differences in peroxisome proliferation which in turn is a result of differences in the rate of formation of TCA from TRI. On this basis it is proposed that TRI presents no significant human hepatocarcinogenic hazard since, human hepatocytes produced TCA at a rate even lower than that of the rat, and TCA was not a peroxisome proliferator in human hepatocytes.