Species Difference In Susceptibility Research Articles

Assessment of metabolic activation of felbamate in chimeric mice with humanized liver in combination with in vitro metabolic assays.

Felbamate (FBM) is an antiepileptic drug that has minimal toxicity in preclinical toxicological species but has a serious idiosyncratic drug toxicity (IDT) in humans. The formation of reactive metabolites is common among most drugs associated with IDT, and 2-phenylpropenal (2-PP) is believed to be the cause of IDT by FBM. It is important to consider the species difference in susceptibility to IDT between experimental animals and humans. In the present study, we used an in vitro and in vivo model system to reveal species difference in IDT of FBM. Human cytochrome P450 (CYP) and carboxylesterase (CES) expressing microsomes were used to clarify the isozymes involved in the metabolism of FBM. The remaining amount of FBM was significantly reduced in incubation with microsomes expressing human CYP2C8, 2C9, 2E1, and CES1c isozymes. Chimeric mice with humanized liver are expected to predict IDT in humans. Therefore, metabolite profiles in chimeric mice with humanized liver were investigated after administration of FBM. Metabolites after glutathione (GSH) conjugation of 2-phenylpropenal (2-PP), which is the reactive metabolite responsible for FBM-induced IDT, were detected in chimeric mice plasma and liver homogenate. Mass spectrometry imaging (MSI) visualizes distribution of FBM and endogenous GSH, and GSH levels in human hepatocyte were decreased after administration of FBM. In this study, we identified CYP and CES isozymes involved in the metabolism of FBM and confirmed reactive metabolite formation and subsequent decrease in GSH using humanized animal model. These results would provide useful information for the susceptibility to IDT between experimental animals and humans.

Lungworm Infection in Small Ruminants in and Around Wolaita Soddo Town, Southern Ethiopia

This study was conducted between November 2010 to April 2011 to estimate the prevalence of lungworm infection in small ruminants, determine the parasite species and identify the potential risk factors in and around Wolaita Soddo town, Southern Ethiopia. Faecal samples were collected from 360 randomly selected small ruminants (204 sheep and 156 goats) and processed with the modified Baerman technique for the extraction of first stage larvae. Overall, 45 (22.1%) sheep and 30 (19.2%) goats were found infected with lungworms. No significant difference was observed in the prevalence of lungworm infection between sheep and goats (p>0.05). Dictyocaulus filaria was the single lungworm species identified in both sheep and goats. The risk factors analyzed were sex, age, management system (zero-grazing or free-ranging), deworming history and body condition score (BCS). Management system, deworming history and BCS were significantly associated with the prevalence of D. filaria infection in sheep and goats (p 0.05). In conclusion, the association of lungworm infections with the management system and deworming status of the animals in the present study are as expected. The association of infection with BCS requires further research to investigate if improving the nutritional status, thereby improving BCS, will result in lower prevalence of lungworm infection. Also, as the present sample size is thought to be small, further study with a larger sample size is required to determine the effect of age and host species difference in susceptibility to lungworm infection. One of the limitations of this study is that it is a one season survey and thus, a further year-round investigation is warranted to establish the association between seasonal variation and infection.

The fate of aflatoxin B1 in animals

The study of the fate of aflatoxin B1 (AFB1) in the rat demonstrated that the main site for the toxin absorption is the small intestine. AFB1 transferred from the intestinal lumen almost exclusively into the mesenteric blood. The increase in the rate of absorption nearly in proportion to the increase in AFB1 concentration in the medium suggests that AFB1 is absorbed substantially by passive diffusion. After being absorbed from the intestine, AFB1 enters exclusively into mesenteric vein and is partially converted to AFL by the blood cell. Comparison of the liver microsomal activity forming AFB1-DNA adduct among various species showed no correlation between the activity and species difference in susceptibility to AFB1 toxicity. However, a reverse relationship was observed between the liver cytosol glutathione-S transferase (GST) activity and species susceptibility, but no relationship was noted between the liver cytosol aldehyde reductase activity toward AFB1 aldehyde activity and species susceptibility. The results indicated the importance of liver cytosol GST activity in species differences in susceptibility to AFB1.

Species difference in 1,3-dinitrobenzene testicular toxicity: In vitro correlation with glutathione status

It has been previously shown that 1,3-dinitrobenzene (DNB) causes testicular damage to the rat but not the hamster. The present study of DNB's mechanism of toxic action has exploited this species difference in susceptibility. Seminiferous tubules were isolated from Golden Syrian hamsters and incubated with 100 μM DNB or vehicle for 22 h. (A similar study with rat tubules has been published.) Formation of DNB metabolites were monitored over time; hamster tubules had a greater capacity than rat tubules for reductively metabolizing (activating) DNB. However, hamster tubules did not show the marked DNB-induced ATP depletion seen in rat tubules. Levels of mitochondrial glutathione and activities of enzymes that protect against oxidative stress were measured in both rat and hamster tubules. The observed differences in the capacity for detoxification of oxidants may underlie the difference in susceptibility to DNB-induced testicular toxicity between these species.

In vitro Aflatoxin B1-DNA Binding by Microsomes and Its Modulation by Cytosol: Comparison of Various Mammalian and Avian Livers in Relation to Species Difference in Susceptibility

Activation and inactivation of aflatoxin B1 (B1) by microsomes and cytosol prepared from the liver of various mammalian and avian species were studied in vitro by determining the microsomal activity to bind aflatoxin to calf thymus DNA and the cytosol activity to inhibit the hamster microsome-mediated aflatoxin-DNA binding. The microsomal activity to bind aflatoxin to DNA was higher in day-old duckling and female chicken than in the other species, being similar in the male hamster, male chicken, both sexes of Japanese quail and laying duck, and lower in the male rat and male mastomys than in the other species. The hamster cytosol inhibited the aflatoxin-DNA binding markedly in the presence of glutathione (GSH) but not at all in its absence. In contrast, the avian cytosol showed a similar level of aflatoxin-DNA binding regardless of the presence or absence of GSH, suggesting that the contribution of cytosol glutathione S-transferase (GST) to B1 detoxification is negligible in the avian species. Nevertheless the cytosol of the avian species such as the Japanese quail and chicken showed apparent inhibitory activity toward aflatoxin-DNA binding. Relative susceptibility of the mammalian and avian species to the toxic and carcinogenic effects of B1 could be explained by the combined activities of microsomes and cytosol, indicating the importance of the opposite activities of microsomes and cytosol toward aflatoxin-DNA binding in the species difference in susceptibility.

Species difference in cholesteryl ester cycle and HDL-induced cholesterol efflux from macrophage foam cells.

The species difference in the turnover rates of the cholesteryl ester (CE) cycle in macrophage foam cells (MFC) was examined in mice and rats. MFC were induced by acetyl-LDL and pulsed with [3H]oleate, followed by a chase with [14C]oleate. The replacement of the initial amount of cholesteryl [3H]oleate by cholesteryl [14C]oleate within 24 hours was 63% in mouse MFC, whereas it was 33% in rat MFC. The corresponding replacement in rabbit MFC was < 10%. In addition, HDL removed 41% of the CE mass from mouse MFC but only 22% from rat MFC. HDL-induced CE reduction from mouse MFC was enhanced by 40% by the inhibitor for acyl-coenzyme A:cholesterol acyltransferase (58-035), whereas the enhancing effect was not observed with rat MFC. These results indicate that the rate of CE turnover may serve as a critical factor to determine the capacity of MFC to respond to HDL-induced CE reduction, suggesting the possibility that the species difference in the turnover rates of the CE cycle in MFC might explain, in part, the species difference in susceptibility to experimental atherosclerosis.

Physiologically Based Toxicokinetic Modeling of 1,3-Butadiene Lung Metabolism in Mice Becomes More Important at Low Doses

This paper describes a physiologically based toxicokinetic model for 1,3-butadiene uptake, distribution, and metabolic clearance in mice. Model parameters for metabolic activity were estimated from the correspondence between computer simulation studies and experimental results as published in the literature. The parameterized model was validated with independent literature data. With the resulting model, the relative importance of lung metabolism as compared to metabolism in the liver increased with decreasing ambient air concentrations. This was due to saturation of metabolism in the alveolar area of the lung, which occurred in the simulations at ambient air concentrations well below current threshold limit values. At higher air concentration, liver metabolism became relatively more important. The tendency toward increased importance of lung metabolism at low doses indicates the necessity of careful extrapolation of in vivo results to low doses. Moreover, this trend may also contribute to species difference in susceptibility to the carcinogenic activity of butadiene.

Physiologically based toxicokinetic modeling of 1,3-butadiene lung metabolism in mice becomes more important at low doses.

This paper describes a physiologically based toxicokinetic model for 1,3-butadiene uptake, distribution, and metabolic clearance in mice. Model parameters for metabolic activity were estimated from the correspondence between computer simulation studies and experimental results as published in the literature. The parameterized model was validated with independent literature data. With the resulting model, the relative importance of lung metabolism as compared to metabolism in the liver increased with decreasing ambient air concentrations. This was due to saturation of metabolism in the alveolar area of the lung, which occurred in the simulations at ambient air concentrations well below current threshold limit values. At higher air concentration, liver metabolism became relatively more important. The tendency toward increased importance of lung metabolism at low doses indicates the necessity of careful extrapolation of in vivo results to low doses. Moreover, this trend may also contribute to species difference in susceptibility to the carcinogenic activity of butadiene.

O-325 - Species difference in susceptibility of egasyn-ß-glucuronidase complex in the liver microsomal membrane to the organophosphates

O-325 - Species difference in susceptibility of egasyn-ß-glucuronidase complex in the liver microsomal membrane to the organophosphates

Species differences in susceptibility to 1,3-dinitrobenzene-induced testicular toxicity and methemoglobinemia

The testicular toxicity and methemoglobinemia induced by 1,3-dinitrobenzene (1,3-DNB) was compared in two species, the Sprague-Dawley rat and the golden Syrian hamster. A marked difference in susceptibility to both endpoints of toxicity was observed. The hamster showed no testicular lesions at dose levels up to 50 mg/kg whereas, as previously reported by others, damage to rat testicular tubules in later stages of spermatogenesis was readily apparent at a 25 mg/kg dose level. Similarly, administration of 1,3-DNB induced substantially less methemoglobinemia in the hamster than in the rat. For example, at the 25 mg/kg dose level peak levels of methemoglobin in the hamster were 15% compared with 80% in the rat. Mortality in the rat also occurred at lower doses than in the hamster (50 vs 100 mg/kg, respectively). In in vitro studies, the capacity of 1,3-DNB and 1,3-DNB metabolites (nitroaniline, nitroacetanilide, aminoacetanilide, diacetamidobenzene) to induce methemoglobinemia was examined in suspensions of red blood cells obtained from both species. Only 1,3-DNB caused the formation of methemoglobin and rat red blood cells were twice as sensitive as hamster red blood cells. The species difference in susceptibility to both methemoglobinemia and testicular toxicity could indicate differences in 1,3-DNB clearance and/or formation of toxic metabolites. Additional metabolic work is under way. This study demonstrates that the hamster is more resistant than the rat to the testicular lesion and methemoglobinemia induced by 1,3-DNB.

Species Differences in Susceptibility to 1,3-Dinitrobenzene-lnduced Testicular Toxicity and Methemoglobinemia

The testicular toxicity and methemoglobinemia induced by 1,3-dinitro-benzene (1,3-DNB) was compared in two species, the Sprague-Dawley rat and the golden Syrian hamster. A marked difference in susceptibility to both endpoints of toxicity was observed. The hamster showed no testicular lesions at dose levels up to 50 mg/kg whereas, as previously reported by others, damage to rat testicular tubules in later stages of spermatogenesis was readily apparent at a 25 mg/kg dose level. Similarly, administration of 1,3-DNB induced substantially less methemoglobinemia in the hamster than in the rat. For example, at the 25 mg/kg dose level peak levels of methemoglobin in the hamster were 15% compared with 80% in the rat. Mortality in the rat also occurred at lower doses than in the hamster (50 vs 100 mg/kg, respectively). In in vitrostudies, the capacity of 1,3-DNB and 1,3-DNB metabolites (nitroaniline, nitroacetanilide, aminoacetanilide, diacetamidobenzene) to induce methemoglobinemia was examined in suspensions of red blood cells obtained from both species. Only 1,3-DNB caused the formation of methemoglobin and rat red blood cells were twice as sensitive as hamster red blood cells. The species difference in susceptibility to both methemoglobinemia and testicular toxicity could indicate differences in 1,3-DNB clearance and/or formation of toxic metabolites. Additional metabolic work is under way. This study demonstrates that the hamster is more resistant than the rat to the testicular lesion and methemoglobinemia induced by 1,3-DNB. c 1991 Society of Toxicoiofy.

Comparison of the disposition and in vitro metabolism of 4-vinylcyclohexene in the female mouse and rat

4-Vinylcyclohexene (VCH) is a chemical to which humans are exposed in the rubber industry. A chronic carcinogenicity bioassay conducted by the National Toxicology Program showed that oral administration of VCH induced tumors in the ovaries of mice but not in those of rats. The hypothesis tested was that the species and organ specificity of VCH toxicity was due to differences in the disposition of VCH between the female rat and mouse. Therefore, the disposition of a single oral dose of 400 mg/kg [ 14C]VCH was studied in female B6C3F1 mice and Fischer 344 rats. Mice eliminated >95% of the dose in 24 hr, whereas rats required 48 hr to eliminate >95% of the dose. The major routes of excretion of [ 14C]VCH-derived radioactivity were in the urine (50–60%) and expired air (30–40%). No evidence was obtained to indicate that the ovaries of either species retained VCH as a parent compound or as radioactive equivalents. A dramatic difference was observed between the rat and mouse in the appearance of a monoepoxide of VCH in blood from 0.5 to 6 hr after VCH administration (800 mg/kg, ip). VCH-1,2-epoxide was present in the blood of mice with the highest concentration at 2 hr (41 nmol/ml). The blood concentration of VCH-1,2-epoxide in rats was <2.5 nmol/ml at all times examined. VCH-7,8-epoxide was not present in the blood of either species at the level of detection. These findings were supported by in vitro studies of VCH epoxidation by liver microsomes. The rate of epoxidation of VCH (1 m m) to VCH-1,2-epoxide was 6.5-fold greater in mouse liver microsomes than that in rat liver microsomes. The species difference in the rate of epoxide formation by the liver may be an important factor in the species difference in susceptibility to VCH-induced ovarian tumors.

Inhalation toxicology of isoprene in F344 rats and B6C3F1 mice following two-week exposures.

Isoprene (2-methyl-1,3-butadiene) was selected for toxicologic evaluations because of its structural similarity to 1,3-butadiene, a potent rodent carcinogen. Two-week inhalation toxicology studies of isoprene were conducted in F344 rats and B6C3F1 mice at exposure concentrations of 0, 438, 875, 1750, 3500, or 7000 ppm. For rats, there were no chemically related changes in survival, body weight gain, clinical signs, hematologic or clinical chemistry parameters, or gross or microscopic lesions. Exposure of mice to isoprene did not produce mortalities and only caused a decrease in body weight gain for male mice in the 7000 ppm exposure group; however, hematologic changes and microscopic lesions including testicular atrophy, olfactory epithelial degeneration, and forestomach epithelial hyperplasia were observed in isoprene-exposed mice. Similar toxicologic effects have been previously observed in B6C3F1 mice exposed to 1,3-butadiene. A species difference in susceptibility between rats and mice exposed to isoprene was evident in these short-term exposure studies.

Effects of trichloroethylene and its metabolites on rodent hepatocyte intercellular communication

Chronic exposure to trichloroethylene (TCE) results in hepatocellular cancer in mice but not rats. The induction of hepatic tumors by TCE appears to be mediated through nongenotoxic or tumor promotion mechanisms. One cellular effect exhibited by a number of nongenotoxic carcinogens and tumor promoters is the inhibition of gap junction mediated intercellular communication. In the present study, the effects of trichloroethylene (TCE) and its metabolites. trichloracetic acid (TCA), trichloroethanol (TCEth), and chloral hydrate (CH) on gap junction mediated intercellular communication in cultured B6C3F1 mouse and F344 rat hepatocytes were assessed. TCE and TCA inhibited intercellular communication in mouse hepatocytes but not in rat hepatocytes. TCEth and CH had no effect on hepatocyte intercellular communication in either rat or mouse cells. TCE and TCA inhibited intercellular communication in both 24-hr-old and freshly plated mouse hepatocytes. Both compounds produced greater inhibition of intercellular communication in freshly plated cells when compared to 24-hr-old cultures. TCE appeared to require cytochrome P450 metabolism by the mouse hepatocytes to exhibit its inhibitory effect on dye coupling since treatment with SKF-525A prevented the inhibition of intercellular communication by TCE. The inhibitory effect of TCA on intercellular communication was unaffected by treatment with SKF-525A. While the species dependent effect of TCE on intercellular communication may be correlated with different rates and extent of metabolism of TCE by rat and mouse hepatocytes, the inhibiting effect of TCA only on mouse hepatocytes suggests that other intrinsic factors in the male mouse make this species more susceptible to the effects of TCE and TCA on gap junction mediated intercellular communication. These findings may account, in part, for the observed species difference in susceptibility to TCE induced liver carcinogenesis.

Differences in ability of NO 2 absorption in various broad-leaved tree species

Absorption of nitrogen dioxide (NO 2) by various broad-leaved tree species was determined by the 15N dilution method. The tree seedlings were continuously exposed to 0·3 ppm (μl litre −1) NO 2 or the mixture of 0·3 ppm NO 2 and 0·1 ppm O 3 for 30 days. The total amount of NO 2-nitrogen absorbed by a seedling during the 30-day exposure period primarily depended on the size of the seedling. Among the tested tree species, three cultivars of Populus showed the highest rate of NO 2 absorption per unit leaf area, reaching as much as 0·3 mg N per dm 2 per day. The absorption rates for Populus cultivars were more than four times greater than those for Viburnum or Cinnamomum which had the lowest rate. A highly significant correlation was recognised between the rate of NO 2 absorption and the stomatal conductance among the species. Three cultivars of Populus which had the highest rates of NO 2 absorption were most susceptible to the mixture of NO 2 and O 3. On the contrary, Cinnamomum, Viburnum and Quercus, which showed the lowest rate of NO 2 absorption, were very tolerant to the mixed gas. These results indicate that the species difference in susceptibility to the mixture of NO 2 and O 3 was mainly determined by the difference in rate of absorption of these gases. Exposure to NO 2 alone had no detrimental effect on the tested tree species.

In-vitro susceptibility testing of Nocardia spp. and its taxonomic implication

By disc diffusion, 28 clinical isolates of four species of Nocardia were tested against 16 antimicrobial agents. Among the drugs included in the study, only a few exhibited in-vitro activities. A species difference in susceptibility was noted for amoxicillin + clavulanic acid, kanamycin, gentamicin, chloramphenicol, minocycline and erythromycin, which was of clinical and taxonomic interest. All six N. brasiliensis and six N. otitidis-caviarum were susceptible to gentamicin and minocycline, while all 15 N. asteroides were not. In addition, N. otitidis-caviarum was susceptible to kanamycin and chloramphenicol, while the sensitivity of N. farcinica was less predictable. The only antibiotic to which members of all of the Nocardia species tested were susceptible was amikacin, which therefore appears to be a good candidate for the treatment of all forms of nocardial infections.

Examination of the differential hepatotoxicity of diallyl phthalate in rats and mice

In this study we confirmed that diallyl phthalate (DAP) is more hepatotoxic to rats than to mice, and we demonstrated the same species difference in toxicity for allyl alcohol (AA). The data suggest that the toxicity of DAP probably results from AA cleaved from DAP. To determine if the species difference in susceptibility to hepatotoxicity resulted from differences in the disposition and metabolism of DAP, Fischer-344 rats and B6C3F1 mice were given [ 14C]DAP, 1, 10, or 100 mg/kg po or 10 mg/kg iv, and placed in metabolism cages for 24 hr. In rats, 25–30% of the DAP was excreted as CO 2, and 50–70% appeared in the urine within 24 hr. In mice, 6–12% of the DAP was excreted as CO 2, and 80–90% was excreted in the urine within 24 hr. Monoallyl phthalate (MAP), allyl alcohol, 3-hydroxypropylmercapturic acid (HPMA), and an unidentified polar metabolite (PM) were found in the urine of rats and mice dosed with DAP. The polar metabolite was present in the urine of rats dosed with DAP or AA, indicating that the compound is a metabolite of AA. There was no difference between the species in the quantity of AA excreted, but mice excreted more MAP (39 vs 33%), HPMA (28 vs 17%), and PM (20 vs 8%) than rats. Because DAP is metabolized to AA, a potent periportal hepatotoxicant, and because the mouse produced more HPMA than rats, we postulate that the differential hepatotoxicity of DAP is related to the extent of glutathione conjugation with allyl alcohol or acrolein (the active metabolite of AA).

Role of hepatic tetrahydrofolate in the species difference in methanol toxicity.

The susceptibility of various species to methanol toxicity is inversely related to the rate of tetrahydrofolate (H4folate)-dependent formate oxidation to carbon dioxide. Thus, the levels of various folate derivatives and folate-dependent enzyme activities present in the livers of monkeys, which are sensitive to methanol, and rats, which are not, were compared in order to investigate the biochemical basis of this species difference. Hepatic H4folate levels in monkeys were 60% of those in rats, and formylated-H4folate derivatives were 2-fold higher in monkeys than in rats. No significant difference between monkeys and rats in the levels of total hepatic folate or 5-methyl-H4folate was observed. The activities of formyl-H4folate synthetase (EC 6.3.4.3) and formyl-H4folate dehydrogenase (EC 1.5.1.6) were 4- and 2-fold higher, respectively, in monkeys than in rats. There was no significant difference between monkeys and rats in methionine synthetase activity (EC 2.1.1.13). Dihydrofolate reductase activity (EC 1.5.1.3) in monkeys was 20% of that in rats. 5,10-Methylene-H4folate reductase (NADPH) activity (EC 1.1.1.171) in monkeys was 40% and 25% of that in rats when the rates of the forward and reverse reactions, respectively, were compared. Serine hydroxymethyltransferase activity (EC 2.1.2.1) was 2-fold higher in monkeys than in rats. The differences in the activities of methylene-H4folate reductase and serine hydroxymethyl-transferase between monkeys and rats may have contributed to the difference in hepatic H4folate levels. The 40% lower level of hepatic H4folate in monkeys, as compared to rats, relates well to the 50% lower maximal rate of formate oxidation in monkeys. Thus, the species difference in susceptibility to methanol may be explained by the difference in the level of hepatic H4folate.

Differences among Nocardia spp. in susceptibility to aminoglycosides and beta-lactam antibiotics and their potential use in taxonomy.

Using disk diffusion and agar dilution methods, 109 clinical isolates of Nocardia were tested against 10 antimicrobial agents. A species difference in susceptibility was noted for cefamandole, carbenicillin, gentamicin, and kanamycin. These differences in susceptibility may be of use taxonomically as well as having some potential use clinically.

Studies on the carcinogenicity of pentachloroethane in rats and mice

The carcinogenicity of chronically administered (41 to 103 weeks) technical-grade pentachloroethane (PCE) was assessed in groups of 50 male and female Fischer 344 rats and B6C3F 1mice. The major contaminant in the PCE sample used for the chronic study was hexachloroethane (4.2%). Prechronic studies (two and 13 weeks repeated dose) employing gavage doses of from 5.0 to 1000 mg/kg/day of PCE failed to reveal specific target organ toxicity in these species. In the absence of treatment-related pathological changes during the prechronic tests, the doses for the chronic studies were set on the basis of survival and body weight gains during the 13-week repeated dose study. During the chronic studies dose levels of 75 and 150 mg/kg for rats and 250 and 500 mg/kg for mice were administered, by gavage, five days per week. The survival of high-dose rats (both sexes) was significantly reduced as compared to the survival of control animals and that of the low-dose males was slightly, but not significantly, reduced. Although the administration of PCE to rats did not increase the incidence of primary tumors, it did produce a significant dose-related increase in the incidence of chronic renal inflammation among males. The survival times of all treated groups of mice were significantly shortened by PCE administration. Despite this reduced survival time, the incidence of hepatocellular carcinoma was significantly increased in all treated groups. With the exception of a dose-related increase in the incidence of hepatocellular adenoma among females, there were no other significant increases in primary tumor incidence in mice. Neither renal lesions in rats nor hepatocellular carcinoma in mice were considered to be adequate explanations for the decreased survivals. Complete histopathological examinations failed to reveal the cause of decreased survival. The results of the study show that technical-grade PCE, like numerous other chlorinated ethanes, is an hepatocarcinogen for B6C3F 1mice. This interpretation for PCE per se, however, must be tempered because the major contaminant, hexachloroethane, has been shown to produce hepatocellular carcinoma in mice. While the results obtained in rats suggest a species difference in susceptibility, the reduced survival in this species may have contributed to the absence of a carcinogenic effect.