Presence Of Exogenous Metabolic Activation Research Articles

Mutagenic and genotoxic in silico QSAR prediction of dimer impurity of gliflozins; canagliflozin, dapaglifozin, and emphagliflozin and in vitro evaluation by Ames and micronucleus test

Canagliflozin, Dapagliflozin, and Empagliflozin, glucagon-like peptide-1 receptor agonists, are indicated for managing type II diabetes. Although the genotoxicity profiles of these drugs are well-explored, limited information exists regarding the genotoxic potential of their impurities. In this investigation, the dimer impurities of Canagliflozin, Dapagliflozin, and Empagliflozin underwent both in silico and in vitro assessments for mutagenic potential. Tester strains of Salmonella typhimurium and Escherichia coli were subjected to the Ames test, utilizing concentrations of up to 1 µg per plate, with and without the presence of metabolic activation. Evaluation of micronucleus induction in TK6 cells was conducted through a micronucleus test, exploring concentrations up to 500 µg/mL, with or without the presence of exogenous metabolic activation. Under the specific test conditions, the dimer impurities of Canagliflozin, Dapagliflozin, and Empagliflozin showed no evidence of mutagenicity or clastrogenicity, establishing their in vitro classification as nonmutagenic. These findings align with negative in silico predictions from quantitative structure–activity relationship (QSAR) analyses for mutagenicity and genotoxicity of the dimer impurities. Collectively, these studies contribute clinically relevant safety information by confirming that the dimer impurities of Canagliflozin, Dapagliflozin, and Empagliflozin are nonmutagenic and nongenotoxic, emphasizing the consistency between in silico and in vitro data.

Human urinary mutagenicity after wood smoke exposure during traditional temazcal use.

In Central America, the traditional temazcales or wood-fired steam baths, commonly used by many Native American populations, are often heated by wood fires with little ventilation, and this use results in high wood smoke exposure. Urinary mutagenicity has been previously employed as a non-invasive biomarker of human exposure to combustion emissions. This study examined the urinary mutagenicity in 19 indigenous Mayan families from the highlands of Guatemala who regularly use temazcales (N = 32), as well as control (unexposed) individuals from the same population (N = 9). Urine samples collected before and after temazcal exposure were enzymatically deconjugated and extracted using solid-phase extraction. The creatinine-adjusted mutagenic potency of urine extracts was assessed using the plate-incorporation version of the Salmonella mutagenicity assay with strain YG1041 in the presence of exogenous metabolic activation. The post-exposure mutagenic potency of urine extracts were, on average, 1.7-fold higher than pre-exposure samples (P < 0.005) and also significantly more mutagenic than the control samples (P < 0.05). Exhaled carbon monoxide (CO) was ~10 times higher following temazcal use (P < 0.0001), and both CO level and time spent in temazcal were positively associated with urinary mutagenic potency (i.e. P < 0.0001 and P = 0.01, respectively). Thus, the wood smoke exposure associated with temazcal use contributes to increased excretion of conjugated mutagenic metabolites. Moreover, urinary mutagenic potency is correlated with other metrics of exposure (i.e. exhaled CO, duration of exposure). Since urinary mutagenicity is a biomarker associated with genetic damage, temazcal use may therefore be expected to contribute to an increased risk of DNA damage and mutation, effects associated with the initiation of cancer.

CYP-450 isoenzymes catalyze the generation of hazardous aromatic amines after reaction with the azo dye Sudan III

This work describes the mutagenic response of Sudan III, an adulterant food dye, using Salmonella typhimurium assay and the generation of hazardous aromatic amines after different oxidation methods of this azo dye. For that, we used metabolic activation by S9, catalytic oxidation by ironporphyrin and electrochemistry oxidation in order to simulate endogenous oxidation conditions. The oxidation reactions promoted discoloration from 65% to 95% of Sudan III at 1 × 10(-4)molL(-1) and generation of 7.6 × 10(-7)molL(-1) to 0.31 × 10(-4)molL(-1) of aniline, o-anisidine, 2-methoxi-5-methylaniline, 4-aminobiphenyl, 4,4'-oxydianiline; 4,4'-diaminodiphenylmethane and 2,6-dimethylaniline. The results were confirmed by LC-MS-MS experiments. We also correlate the mutagenic effects of Sudan III using S. typhimurium with the strain TA1535 in the presence of exogenous metabolic activation (S9) with the metabolization products of this compound. Our findings clearly indicate that aromatic amines are formed due to oxidative reactions that can be promoted by hepatic cells, after the ingestion of Sudan III. Considering that, the use of azo compounds as food dyestuffs should be carefully controlled.

Mutagenic assessment of Prestige fuel oil spilled on the shore and submitted to field trials of bioremediation

The mutagenicy of the slightly weathered fuel oil from the Prestige oil spill and the effects of different bioremediation products (nutrients and/or microorganisms and biodiesel) on the potential mutagenic activity of this heavy fuel oil spilled on the shore were evaluated for a period of 1 year using the Ames Salmonella assay with strains TA98, TA100, TA1535 and TA1537 in the absence and presence of exogenous metabolic activation (S9 fraction from rat liver). The in situ bioremediation experiment was performed using tiles located in the supra-littoral and intertidal zones of a beach seriously affected by the fuel oil spill. The results obtained showed the mutagenic activity of the slightly weathered fuel oil extracts at the beginning of the experiment in strain TA98 that persisted for more than 150 days in both the untreated control and treated tiles independently of the zone of the beach considered. However, after 360 days neither the control nor the treated tiles in the intertidal zone showed mutagenic activity and a weak positive response in strain TA98 was detected for the control fuel oil extracts from supra-littoral tiles. The application of biodiesel to accelerate the biodegradation of this type of fuel oil may constitute a further genotoxic hazard to the environment, since the mutagenic response achieved from the biodiesel–fuel oil mixture in the first samplings (days 0 and 30) was more potent than that obtained from the control tiles. The mutagenic activity was detected along the study with S. typhimurium TA98 in both the presence and absence of S9 microsomal fraction, but the addition of S9 fraction in the assay always increased the number of revertants induced. In general, these findings suggest that the bioremediation strategies used were not effective in eliminating the genotoxic hazard associated with this heavy fuel oil attached to rocky substrate since they did not achieve a decrease in the mutagenic response with respect to the untreated control tiles. These data also confirm that genotoxicity assays should be used to evaluate the effectiveness of bioremediation efforts associated with oil spills for a better risk assessment.

Evaluation of the Mutagenic and Genotoxic Potential of Ubiquinol

Ubiquinol (the reduced form of coenzyme Q(10)) is the two-electron reduction product of ubiquinone (the oxidized form of coenzyme Q(10)), and has been shown to be an integral part of living cells, where it functions as an antioxidant in both mitochondria and lipid membranes. To provide information to enable a Generally Regarded as Safe (GRAS) evaluation for the use of ubiquinol in selected foods, a series of Organisation of Economic Cooperation and Development (OECD) and good laboratory practice (GLP) toxicological studies was conducted to evaluate the mutagenic and genotoxic potential of Kaneka QH brand of ubiquinol. Ubiquinol did not induce reverse mutations in Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA at concentrations up to 5000 mu g/plate, in either the absence and presence of exogenous metabolic activation by rat liver S9. Likewise, ubiquinol did not induce chromosome aberrations in Chinese hamster lung fibroblast (CHL/IU) cells in short-term (6-h) tests with or without rat liver S9 at concentrations up to 5000 mu g/ml or in a continuous (24-h) treatment test at concentrations up to 1201 mu g/ml. Finally, no mortalities, no abnormal clinical signs, and no significant increase in chromosome damage were observed in an in vivo micronucleus test when administered orally at doses up to 2000 mg/kg/day. Thus, ubiquinol was evaluated as negative in the bacterial reverse mutation, chromosomal aberration, and rat bone marrow micronucleus tests under the conditions of these assays.

Absence of Mutagenicity, Genotoxicity, and Subchronic Oral Toxicity of Touchi Extract

Touchi, a traditional Chinese food used mainly for seasoning is obtained by first steaming soybeans followed by fermentation with Aspergillus oryzae (koji). A series of toxicological studies was conducted to evaluate the mutagenic and genotoxic potential and subchronic toxicity of a water extract of Touchi, a known inhibitor of alpha -glucosidase activity. Touchi extract (TE) did not induce reverse mutations in Salmonella typhimurium strains TA98, TA1537, TA100, TA1535, and Escherichia coli WP2uvrA at concentrations up to 5000 microg/plate, in either the absence or presence of exogenous metabolic activation. No deaths occurred and no abnormal clinical signs were observed in any animal in any group in an in vivo micronucleus test, and TE was devoid of clastogenic activity when administered orally to mice at doses up to 2000 mg/kg/day. Thus, TE was evaluated as negative in the bacterial reverse mutation and mouse bone marrow micronucleus tests under the conditions of these assays. To evaluate its subchronic toxicity, SPF rats were administered TE at doses of 0, 250, 1000, and 2500 mg/kg/day via oral gastric intubation. No treatment-related toxic changes were seen in clinical signs, body weight, food consumption, urinalysis, hematology, blood chemistry, necropsy, organ weight, or histopathology. The no observed adverse effect level for TE was thus considered to be more than 2500 mg/kg/day in both males and females. These results are consistent with Touchi's status as a traditional Chinese food derived from fermented soybeans and its purported long history of use. Specifically, these data are consistent with the expected safety of human consumption of TE up to at least 5 g/day.

Ability of root canal antiseptics used in dental practice to induce chromosome aberrations in human dental pulp cells

Root canal antiseptics are topically applied to root canals within the pulpless teeth to treat the root canal and periapical infections. Because the antiseptics that are applied to root canals can penetrate through dentin or leak out through an apical foramen into the periodontium and distribute by the systemic circulation, it is important to study the safety of these antiseptics. In the present study, we examined the ability to induce chromosome aberrations in human dental pulp cells of five root canal antiseptics, namely, carbol camphor (CC), camphorated p-monochlorophenol (CMCP), formocresol (FC), calcium hydroxide, and iodoform which are most commonly used in dental practice. Statistically significant increases in the levels of chromosome aberrations were induced by CC, FC, or iodoform in a concentration-dependent manner. Conversely, CMCP and calcium hydroxide failed to induce chromosome aberrations in the absence or presence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were enhanced by FC or iodoform. Our results indicate that the root canal antiseptics that exhibited a positive response are potentially genotoxic to human cells.

In vitro chromosome aberration tests using human dental pulp cells to detect the carcinogenic potential of chemical agents

To examine if human dental pulp cells are useful for assessing the carcinogenic potential of chemical agents, we cultured human dental pulp cells from adults and studied the ability of chemical agents known to be carcinogenic to induce chromosome aberrations in these cells. We confirmed that human dental pulp cells in primary or secondary cultures had the capability of accumulating calcium in vitro as detected by Alizarin red staining and generating dentin-like tissue in immunocompromised mice. These phenotypes were maintained even in cells at seven passages. Next, we examined if chromosome aberrations were induced by exposure of human dental pulp cells (designated here as D824 cells) at seven to nine passages to chemical agents with carcinogenic activity. Statistically significant increases in the frequencies of chromosome aberrations were induced in D824 cells treated with a direct-acting carcinogen, mitomycin C, for 3 h. Chromosome aberrations were also induced at statistically significant levels in D824 cells treated with an indirect-acting carcinogen, cyclophosphamide, for 2 h in the presence of exogenous metabolic activation with rat liver postmitochondrial supernatant. Cyclophosphamide failed to induce chromosome aberrations in the absence of exogenous metabolic activation. Although the reliability of chromosome aberration tests using human dental pulp cells remains to be validated by studying the ability of various other chemical agents with or without carcinogenic activity to induce chromosome aberrations, this chromosome aberration test system may be useful for carcinogenic risk assessment in the target cells.

Induction of Micronuclei in Human TK6 Lymphoblasts by 2‐Dodecylcyclobutanone, a Unique Radiolytic Product of Palmitic Acid

ABSTRACT: Palmitic acid, the most abundant fatty acid in the human diet, causes oxidative DNA damage, DNA strand breakage, necrosis, and apoptosis in human and rodent cells in vitro, while excess fatty acids in the diet increase the risk of colon cancer in vivo. Irradiation of palmitic acid leads to the formation of 2‐dodecylcyclobutanone (2‐DCB), a unique radiolytic product formed at part‐per‐million levels in fats. Recent research has raised the possibility that, like palmitic acid, 2‐DCB can also cause DNA strand breaks in human and rodent cells. In order to more fully understand the clastogenic potential of 2‐DCB, it was tested for the ability to induce the formation of micronuclei (MN) in human TK6 lymphoblasts. TK6 lymphoblasts were exposed to 13.3, 26.5, and 53 μM of 2‐DCB for 24 h, both with and without exogenous metabolic activation (EMA). The number of MN per 1000 binucleated cells was induced 2.65‐fold without EMA, and 2.85‐fold in the presence of EMA at the highest 2‐DCB concentration of 53 μM. Cytotoxicity of the TK6 lymphoblasts treated with 53 μM 2‐DCB was 51.7% in the absence of EMA, and 61.7% in the presence of EMA. Thus, like palmitic acid, 2‐DCB may be weakly clastogenic against human cells in vitro. Given the amount of 2‐DCB that would be consumed when compared with that of its clastogenic parent fatty acid, 2‐DCB is unlikely to have any significant impact on human health.

Assessment of genotoxicity of 14 chemical agents used in dental practice: Ability to induce chromosome aberrations in Syrian hamster embryo cells

To assess the genotoxicity of 14 chemical agents used as locally applied agents in dental practice, the ability of these agents to elicit chromosome aberrations was examined using Syrian hamster embryo (SHE) cells. Chromosome aberrations in SHE cells were induced by treatment with three of eight chemical agents used as endodontic medicaments, i.e. ethylenediaminetetraacetic acid (EDTA), formocresol (a mixture of formalin and tricresol), and sodium arsenite. The other five chemical agents, i.e. chloramphenicol, p-chlorophenol, p-phenolsulfonic acid, sodium hypochlorite, and tetracycline hydrochloride exhibited a negative response for chromosome aberrations. Assessment of three dyes used for disclosing dental plaque showed chromosome aberrations induced by basic fuchsin but not by acid fuchsin and erythrosine B. Three local anesthetics, lidocaine hydrochloride, prilocaine hydrochloride, and procaine hydrochloride, were negative for chromosome aberrations. Among the ten chemical agents that exhibited a negative response in the assay, p-chlorophenol, sodium hypochlorite, and erythrosine B induced chromosome aberrations in SHE cells when treated in the presence of exogenous metabolic activation. The percentages of cells with polyploidy or endoreduplication were enhanced by formocresol, sodium arsenite, p-chlorophenol, p-phenolsulfonic acid, sodium hypochlorite, erythrosine B, prilocaine hydrochloride, and procaine hydrochloride in the absence or presence of exogenous metabolic activation. Our results indicate that the chemical agents that had a positive response in the present study are potentially genotoxic to mammalian cells.

Ability of Fourteen Chemical Agents Used in Dental Practice to Induce Chromosome Aberrations in Syrian Hamster Embryo Cells

To assess the genotoxicity of 14 chemical agents used in dental practice, the ability of these agents to induce chromosome aberrations was examined using Syrian hamster embryo (SHE) cells. Statistically significant increases in the frequencies of chromosome aberrations were induced in SHE cells treated with 7 of 10 chemical agents used as endodontic medicaments, that is, carbol camphor, m-cresol, eugenol, guaiacol, zinc oxide, hydrogen peroxide, and formaldehyde. The other 3 chemical agents, that is, thymol, glutaraldehyde, and iodoform, did not increase the levels of chromosome aberrations. Of the 4 chemical agents that are used as an antiseptic on the oral mucosa, chromosome aberrations were induced by iodine, but not by the other 3 antiseptics, benzalkonium chloride, benzethonium chloride, and chlorhexidine. Among the 6 chemical agents exhibiting a negative response in the assay, only thymol induced chromosome aberrations in the presence of exogenous metabolic activation. Our results indicate that chemical agents having a positive response in the present study are potentially genotoxic to mammalian cells and need to be studied further in detail.

Lack of clastogenic effects in cultured human lymphocytes treated with hydroquinone

Hydroquinone (HQ) occurs in the environment as a result of manmade processes as well as in natural products from plants and animals. The compound has been reported to produce chromosomal effects in some in vivo and in vitro animal models. However, its potential to produce similar effects in human lymphocytes is less clear. To obtain more information on the clastogenic potential of HQ in human cells, its ability to induce structural chromosomal aberrations in human lymphocytes in vitro has been examined, both in the absence and presence of exogenous metabolic activation. Moreover, the effect of HQ pre-incubation on peroxide induced clastogenicity was studied, because HQ has putative chemopreventive activity as well. It was found that HQ was cytotoxic, but did not induce chromosomal aberrations in human lymphocytes cultured in vitro. Additionally, it was observed that pre-incubation of lymphocytes with HQ resulted in a concentration dependent reduction of the H 2O 2 induced chromosomal aberrations ( P=0.069). However, this effect was present at 12 mM H 2O 2 only, because of high cytotoxicity at higher dosages.

Cytological and cytogenetic effects of the fluoroquinolone ofloxacin on human periodontal ligament fibroblasts.

The fluoroquinolone ofloxacin (OFLX) is one of the candidates of antibacterial agents to be topically used against periodontitis. To estimate the maximum concentration of OFLX which exerts little or no adverse effect on the periodontal ligament, cytological and cytogenetic effects of OFLX on human periodontal ligament fibroblasts (Pel cells) were examined. Treatment of Pel cells with < or =0.3 mM OFLX for 24 or 48 h had little inhibitory effect on cellular growth and survival. DNA, RNA and protein syntheses in Pel cells did not decrease in response to treatment with < or =0.3 mM OFLX. The constitutive level of alkaline phosphatase mRNA was retained in cells treated with < or = 0.03 mM OFLX for 24 or 48 h. The level of type I procollagen mRNA was not affected by treatment with < or = 0.003 mM OFLX for 24 or 48 h. Cytogenetic effects of OFLX were evaluated by the ability of OFLX to induce chromosome aberrations in Pel cells. Treatment with OFLX at 0.3-3.0 mM for 6, 24, or 48 h failed to induce chromosome aberrations in Pel cells. The failure of OFLX to induce chromosome aberrations was seen even in the presence of exogenous metabolic activation using a 5% rat liver post-mitochondrial supernatant mixture. These results indicate that treatment of Pel cells with < or =0.003 mM OFLX has few or no adverse effects on the cytological and cytogenetic endpoints examined, suggesting that there would be little adverse effect on growth and differentiation of the periodontal ligament, as well as little cytogenetic activity, if OFLX were to be topically administered to the gingival crevice at the minimal inhibitory concentration (MIC90) against periodontopathic bacteria (< or = 0.0027 mM). It is important to note, however, that extrapolation of these findings to in vivo conditions has yet to be undertaken.

Assessment of genotoxicity of dental antiseptics: ability of phenol, guaiacol, p-phenolsulfonic acid, sodium hypochlorite, p-chlorophenol, m-cresol or formaldehyde to induce unscheduled DNA synthesis in cultured Syrian hamster embryo cells.

To assess the genotoxicity of seven dental antiseptics, the ability of these agents to induce unscheduled DNA synthesis (UDS) was examined using Syrian hamster embryo (SHE) cells. Treatment of SHE cells with phenol or formaldehyde induced UDS in a concentration-dependent manner as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis. Guaiacol or m-cresol induced UDS only in the presence of exogenous metabolic activation. p-Phenolsulfonic acid, sodium hypochlorite and p-chlorophenol failed to induce UDS in the presence or absence of exogenous metabolic activation. Our results suggest that phenol, guaiacol, m-cresol and formaldehyde are genotoxic to mammalian cells.

Estrogen-induced cell transformation and DNA adduct formation in cultured Syrian hamster embryo cells.

To study the possible involvement of DNA damage in cell transformation induced by estrogens, we examined whether DNA adduct formation is elicited in cultured Syrian hamster embryo (SHE) cells treated with estrogens and their derivatives by means of the 32P-postlabeling assay. Morphological transformation of the cells was induced by treatment with diethylstilbestrol (DES) at 1-10 micrograms/mL for 24 h but not by treatment with its derivatives trans, trans-dienestrol (alpha-DIES) or cis, cis-dienestrol (beta-DIES) at 1-10 micrograms/mL for 24 h. Similarly, DNA adduct formation was elicited by exposure of SHE cells to DES at 1-10 micrograms/mL for 24 h but not by either alpha-DIES or beta-DIES. Treatment of SHE cells with DES at 1-10 micrograms/mL for 2 h in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant enhanced morphological transformation in a dose-dependent manner. Our previous studies have demonstrated that exposure of SHE cells to DES under the same conditions with exogenous metabolic activation induces somatic mutations at the Na+/K+ ATPase locus. Therefore, we examined whether with exogenous metabolic activation DES induced DNA adduct formation in SHE cells. DNA adducts were not detected when SHE cells were treated with DES at 1-10 micrograms/mL for 2 h in the presence of exogenous metabolic activation. Treatment with 17 beta-estradiol (E2), 2-hydroxyestradiol (2-OH E2), or 4-hydroxyestradiol (4-OH E2) at 1 microgram/mL for 24 h induced DNA adduct formation in the cells, in parallel with the induction of cell transformation. The rank order of DNA adduct formation was 4-OH E2 > 2-OH E2 > E2. The results indicate that estrogens induce DNA adduct formation in cultured SHE cells, but the induction may not be the only mechanism relevant to the initiation of cell transformation.

Treatment with aflatoxin B1 for immortalization of human fibroblasts from Li-Fraumeni patients

Immortalization of normal human fibroblasts is a very rare event. Multiple genes such as p53 and cellular senescence genes are possibly involved in immortalization of human fibroblasts, suggesting that multiple treatments with carcinogens are required for the immortalization. We describe here the procedure for immortalization of human fibroblasts (MDAH 087) from Li-Fraumeni cancer syndrome with a germ-line p53 mutation. The cells were subjected to multiple treatments with aflatoxin B1 (AFB1) in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), and 3 of 9 MDAH 087 cell cultures treated 1–3 times with 0.1–1 µg/ml AFB1 became immortal, defined as continuous growth for over 300 population doublings after the first treatment. However, cultures of human fibroblasts from a normal embryo treated under the same conditions failed to escape senescence. The results indicate that the model of human fibroblasts with a mutated p53 allele exposed to AFB1 is potentially useful for studying mechanisms of chemically induced immortalization.

Maternal smoking induced cotinine levels and genotoxicity in second trimester amniotic fluid

Cotinine concentrations in amniotic fluid samples from 22 smoking and 37 non-smoking pregnant women and induction of sister-chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells by samples from 15 smokers and 15 non-smokers were studied as indicators of exposure to potential genotoxic activity during pregnancy. Analysis of cotinine revealed one individual in the non-smoking group with a high cotinine level apparently due to non-reported smoking. The mean cotinine concentration of smokers was 85 ng/ml whereas non-smokers had a concentration of 0.3 ng/ml. According to interview data 16 persons announced some passive exposure to tobacco smoke at home or at work; however this group did not differ from unexposed non-smokers in their amniotic fluid cotinine concentration. SCE inducing activity was tested with and without metabolic activation. The mean SCE frequency in CHO cells induced in the presence of exogenous metabolic activation by concentrated amniotic fluid of heavy smokers ( ≧ 10 cigarettes/day ) was significantly higher (9.7±0.6 SCE/cell) than among non-smokers (8.9±0.6 SCE/cell) with metabolic activation. The results show that amniotic fluid cotinine measurements and induction of SCEs in CHO cells can be used to indicate fetal exposure by maternal smoking and support earlier studies suggesting a potential genotoxic hazard to the fetus of heavy smokers.

Ability of peroxisome proliferators to induce cell transformation, chromosome aberrations and peroxisome proliferation in cultured Syrian hamster embryo cells.

Di(2-ethylhexyl)phthalate (DEHP), a commonly used plasticizer, induces proliferation of peroxisomes in liver cells and causes hepatocellular carcinomas when chronically administered in the diet to rodents. To examine possible mechanisms for DEHP-associated cancer, we have measured induction of morphological transformation, chromosome aberrations and peroxisome proliferations of cultured Syrian hamster embryo (SHE) cells by DEHP and other peroxisome proliferators. Morphological transformation of SHE cells was weakly induced by treatment for 48 h with DEHP and its metabolite mono(2-ethylhexyl)phthalate (MEHP). The transformation frequency by DEHP was enhanced by exogenous metabolic activation using rat liver postmitochondrial supernatants. Treatment for 24 h with DEHP resulted in chromosome aberrations of the cells only in the presence of exogenous metabolic activation. 2-(p-chlorophenoxy)-2-methylpropionic acid ethyl ester (clofibrate), a widely used hypolipidemic drug, failed to induce morphological transformation or chromosome aberrations of SHE cells. Treatment with [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY-14,643), which is a more potent carcinogen than DEHP or clofibrate, elicited a lower frequency of morphological transformation than DEHP in the presence of exogenous metabolic activation but was more active than DEHP at inducing chromosome aberrations. Similar levels of peroxisome proliferation, as determined by an intensity of diaminobenzidine staining, were observed in cultures treated for 2 h with DEHP, MEHP, clofibrate or WY-14,643. These results suggest a possible involvement of genetic damage by DEHP metabolites in the induction of cell transformation of SHE cells by DEHP; however, no clear relationship among induction of peroxisome proliferation, carcinogenicity in vivo and cell transformation was observed. Although the ability to induce cell transformation and chromosomal mutations is not adequate to explain the carcinogenicity of this class of compounds, these biological effects may be contributory to the carcinogenic activities of peroxisome proliferators.

Mutagenic and clastogenic properties of (3-[3-(6-benzoyloxy-3-cyano-2- pyridyloxycarbonyl) benzoyl]-1-ethoxymethyl-5-fluorouracil) (BOF-A2), a new 5-fluorouracil derivative with anti-tumour activity.

As part of the safety assessment of (3-[3-(6-benzoyloxy-3-cyano-2- pyridyloxycarbonyl)benzoyl]-1-ethoxymethyl-5-fluorouracil) (BOF-A2), a new 5-fluorouracil (5-FU) derivative with anti-tumour activity, its potential genotoxicity was studied in 3 different tests. BOF-A2 was negative in a reverse mutation (Ames) test in strains of S. typhimurium and E. coli. BOF-A2 induced chromosomal aberrations in Chinese hamster cells in vitro especially in the presence of exogenous metabolic activation, and was clastogenic in vivo, inducing micronuclei in mouse bone marrow. The clastogenic activity of BOF-A2 was similar to that of 5-FU.

Evaluation of genotoxic effects of the herbicide dicamba using in vivo and in vitro test systems

The genotoxic effects of the herbicide dicamba have been studied by measuring 1) the unwinding rate of liver DNA from intraperitoneally (i.p.) treated rats (fluorimetric assay); 2) DNA repair as unscheduled DNA synthesis (UDS) induced in cultured human peripheral blood lymphocytes (HPBL); and 3) sister chromatid exchanges (SCE) in HPBL. Results show that dicamba is capable of inducing DNA damage since it significantly increases the unwinding rate of rat liver DNA in vivo and also induces UDS in HPBL in vitro in the presence of exogenous metabolic activation (S-9 mix). Furthermore, dicamba causes a very slight increase in SCE frequency in HPBL in vitro.