Genetic and metabolic effects of gluconasturtiin, a glucosinolate derived from cruciferae.

It is thought that induction of detoxifying phase-II drug metabolizing enzymes or inhibition of bioactivating phase-I by phytoalexins could protect against mutagens and neoplasia. In the search for potential naturally occurring molecular chemoprevention agents, particular attention has been devoted to isothiocyanates, which are breakdown products-via myrosinase-of glucosinolates such as gluconasturtiin (GNST), a natural constituent of cruciferae. Here, we first investigated the ability of GNST to modulate metabolizing enzymes in male Swiss Albino CD1 mice injected by gavage (24 mg/kg or 48 mg/kg b.w.) with GNST either in single or repeated (daily for four consecutive days) dose. Using selected probes to various cytochrome P450 (CYP) isoforms, a marked and generalized decrease of CYP content, NADPH-(CYP)-c-reductase and various CYP-linked monooxygenases (measuring CYP1A1, CYP2B1/2, CYP3A1/2, CYP1A2 and CYP2E1), was observed in hepatic, renal and pulmonary subcellular preparations (up to approximately 66% loss, liver). Similar behavior was recorded using the regio- and stereo-selective hydroxylation of testosterone as multibiomarker (CYP2A1 and CYP2B9, up to approximately 96% loss), as well as with the phase-II marker glutathione S-transferase (up to approximately 50% loss, liver). We also performed genotoxicity investigations, using the diploid D7 strain of yeast Saccharomyces cerevisiae as a biological test system. GNST was able to significantly induce point reverse mutation in growing cells without myrosinase, thus suggesting either a direct GNST or a CYP-linked metabolite role in the genotoxic response. On the contrary, in suspension test, the addition of myrosinase significantly increased mitotic gene conversion, probably due to the formation of GNST-derived phenylethyl isothiocyanate (PEITC) breakdown product. Taken together, our data suggest that GNST exerts a dual effect: while strongly inhibiting the microsomal (bioactivating) metabolism, GNST also possesses genotoxic activity. This concomitant mutagenic activity underlines the necessity of overall toxicological characterization of this (or any other molecule) prior to mass chemopreventive use.

Induction of cytochrome P450 enzymes and over-generation of oxygen radicals in beta-carotene supplemented rats.

Effects of beta-carotene (betaCT) on microsomal CYP-linked monooxygenases were investigated using both the regio- and stereo-selective hydroxylation of testosterone (as multibiomarker) and highly specific substrates as probes of various isoenzymes. CYP-catalyzed reactions were studied in the liver, kidney, lung and intestine of Sprague-Dawley rats of both sexes supplemented with 250 or 500 mg/kg body wt betaCT (per os) in a single or repeated (daily for 5 days) fashion. Generalized boosting effects (2-15-fold increases) were observed in the various tissues for carcinogen metabolizing enzymes associated with CYP1A1/2, CYP3A1/2, CYP2E1, CYP2B1/2 and CYP2C11. Induction of the most affected CYPs was corroborated by western blot linked to densitometric analyses. Measurement of reactive oxygen species (ROS) produced by subcellular preparations from either control or betaCT supplemented rats was performed by EPR detection of the nitroxide radical yielded by the reaction with ROS of the hydroxylamine spin probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate. Marked ROS over-generation associated with CYP induction (up to 33-fold increase in the liver) was recorded in the various organs (liver > lung > intestine > kidney). CYP and ROS induction are substantially in keeping with the concentration of betaCT accumulated in the various tissues, the liver being the most affected organ. These findings are consistent with the concept that betaCT is a pro-oxidant and potentially co-carcinogenic pro-vitamin, and may help explain why, in large quantities, it can have harmful effects in humans.

Inhibitory activity of vitamin E and alpha-naphthoflavone on beta-carotene-enhanced transformation of BALB/c 3T3 cells by benzo(a)pyrene and cigarette-smoke condensate.

We previously found that beta-carotene (betaCT) can act as a co-carcinogenic agent enhancing the cell transforming activity of powerful carcinogens such as benzo(a)pyrene (B(a)P) and cigarette-smoke condensate (TAR) in an in vitro medium-term ( approximately 8 weeks) experimental model utilizing BALB/c 3T3 cells (Mutat. Res. 440 (1999) 83-90). Here, we investigated whether vitamin E (VitE) and alpha-naphthoflavone (alphaNF) are able to affect the co-carcinogenic activity of betaCT in terms of inhibiting B(a)P and TAR cell transforming potential. The following experimental schedules were performed: (i) cultures treated for 72 h with chemicals in various experimental combinations (acute treatment); (ii) cultures grown in presence of tester agents for the whole period of the assay (chronic treatment) to more closely mimic human exposure. While the co-carcinogenic potential of betaCT was confirmed on both B(a)P and TAR, the latter being ineffective by itself, we found in repeated experiments that the presence of VitE or alphaNF significantly reduced the betaCT's enhancing effect in the formation of transformation foci by B(a)P and TAR. The mechanism of the inhibition could be explained by the known ability of alphaNF to inhibit cytochrome P450-linked B(a)P-bioactivating monooxygenases, while VitE may contrast the prooxidant activity of betaCT (e.g., oxygen radicals overgeneration). While highlighting the importance of increasing knowledge of the role of single provitamins, vitamins and micronutrients, our findings also underline the potential advantages of combining several dietary supplements in in vitro preventive investigations.

Effect of liquorice and glycyrrhizin on rat liver carcinogen metabolizing enzymes.

We investigated the effect of single or repeated intake of conspicuous amounts of licorice root extract (LE, 3138 or 6276 mg/kg body weight (bw) per os) or its natural constituent glycyrrhizin (G, 240 or 480 mg/kg bw per os) on Sprague-Dawley rat liver monooxygenases. Whereas a single LE or G dose was unable to affect CYP superfamily, four daily doses induced CYP3A, CYP1A2 and to varying extents CYP2B1-linked monooxygenases. A boosting effect on testosterone 6beta- (CYP3A1/2, CYP1A1/2), 7alpha- (CYP1A1/2, CYP2A1), 16alpha- (CYP2B1, CYP2C11), 2alpha- (CYP2C11) and 2beta- (CYP3A1, CYP1A1) -dependent oxidases as well as on androst-4-ene-3,17-dione- (CYP3A1/2) -supported monooxygenases were also achieved. Harmful outcomes associated to CYP changes (e.g. cotoxicity, cocarcinogenicity and promotion) may be of concern.

beta-carotene as enhancer of cell transforming activity of powerful carcinogens and cigarette-smoke condensate on BALB/c 3T3 cells in vitro.

We report the ability of beta-carotene (betaC) to affect the cell transforming activity of 3-methylcholanthrene (3-MCA), benzo(a)pyrene (B(a)P) and cigarette-smoke condensate (TAR) in an in vitro medium-term (approximately 8 weeks) experimental model utilizing BALB/c 3T3 cells. Different experimental schedules were performed either in the presence or absence of betaC: (i) cultures treated for 72 h with each chemical (acute treatment), (ii) cultures grown in presence of each chemical for the whole period of the experiment (chronic treatment). These procedures suggested a possible cocarcinogenic potential of the carotenoid following interactions with other chemicals mimicking continuous human exposition to several xenobiotics. Although the pigment did not show any cell transforming potential when tested alone either in acute or chronic treatment, it did augment that of other tested agents. Induction of cell transformation by B(a)P was markedly enhanced by the presence of this carotenoid in either acute or chronic treatment. Only in presence of betaC, was TAR able to significantly act as a cell transforming agent in prolonged, chronic treatment of cultures. Enhanced cell transformation activity could be due to the boosting effect of betaC on P450 apparatus. Indeed, elsewhere we have found that the latter increased the ratio of formation of diol epoxide carcinogenic metabolites of B(a)P as well as other carcinogens present in TAR. By contrast, no differences of cell transforming activity of 3-MCA, an ultimate carcinogen, were seen either in the presence or absence of betaC under the various experimental conditions. These data, which are in keeping with the cocarcinogenic potential of betaC, may help to explain the unexpected lung cancer increases obtained in chemoprevention trials in heavy smokers supplemented with the isoprenoid. Our findings also highlight the potential risk to humans derived from interactions among xenobiotics present in the environment.

Molecular non-genetic biomarkers of effect related to methyl thiophanate cocarcinogenesis: organ- and sex-specific cytochrome P450 induction in the rat.

We used selective biochemical markers of effect to evaluate some non-genotoxic cocarcinogenic properties of methyl thiophanate (MTH) associated with cytochrome P450 (CYP) changes. Several CYP-dependent reactions were monitored in the liver, kidney and lung microsomes of male and female Sprague-Dawley rats treated (i.p.) with a single (285 or 570 mg/kg body weight) or repeated (daily 285 or 570 mg/kg body weight for three consecutive days) doses of this pesticide. No significant changes in absolute or relative liver, kidney and lung weights were observed after MTH injection. Highly specific substrates were used as probes of different isoforms, such as CYP1A1, 1A2, 2B1, 2E1 and 3A. A complex pattern of CYP induction, including organ- and sex-related differences, was observed, particularly in the liver (CYP3A, 2B1), kidney (CYP1A1, 2E1) and lung (CYP3A, 1A1). In the liver, an increase up to 29-fold in the 2B1-like activity, probed by the O-dealkylation of pentoxyresorufin, was observed at lower dose in both sexes, and the induction of CYP 1A2-mediated methoxyresorufin O-demethylase activity (up to 3.6-fold) was recorded at the higher dose in males. In the kidney, the O-deethylation of ethoxyresorufin (CYP1A1-linked) was increased up to 28.2-fold and the CYP2E1-dependent p-nitrophenol hydroxylases were enhanced up to 6.3-fold in females receiving higher multiple MTH administration. In the lung, the CYP3A-associated activity was the most induced oxidases, as exemplified by the marked increase in the O-demethylation of aminopyrine (up to 3.6-fold) in males. A weak, although significant, reduction of CYP2B1-linked oxidases was also observed in repeated treatment in the kidney (males) and lung (females). These results suggest that the induction of CYP-catalyzed drug metabolism by prolonged exposure to MTH may result in accelerated metabolism of coadministered drugs with important implications for their disposition Together with an alteration of endogenous metabolism, the adverse effects associated with CYP changes such as toxicity/cotoxicity, cocarcinogenicity and promotion may also have clinical consequences.

Effect of butyrate analogues on proliferation and differentiation in human neuroblastoma cell lines.

Butyric acid has been shown in vitro to produce cytodifferentiation of a wide variety of neoplastic cells. The potential clinical use of this compound as a therapeutic agent is limited by its rapid metabolism. This has led to the examination, as potential antineoplastic agents, of compounds structurally correlated to butyrate, with longer biological half lives. In this study we investigated the effect in vitro of two butyrate analogues, tributyrin and butyramide, on inducing growth inhibition and expression of morphological and immunophenotypic properties, in human neuroblastoma cell lines. Treatment with tributyrin resulted in a strong inhibition of cell proliferation and in induction of extensive differentiation; on the contrary butyramide was scarcely effective or quite ineffective. These results demonstrate that tributyrin retains the effectiveness of butyrate and suggest that this analogue could have utility for cytodifferentiation therapy.

Molecular non-genetic biomarkers of effect related to acephate cocarcinogenesis: sex- and tissue-dependent induction or suppression of murine CYPs.

The aim of this work was to study the ability of the organophosphate insecticide acephate to alter some biochemical markers of effect related non-genetic cocarcinogenesis. For this purpose, selective CYP-dependent reactions have been examined in liver, kidney and lung microsomes of male and female Swiss albino CD1-mice treated (i.p.) with a 125 or 250 mg/kg b.w. dose of this pesticide. High specific substrates were used as a probe of various isozymes, such as CYP 1A1, 1A2, 2B1, 2E1 and 3A. Maked organ- and sex-related differences in either inducive or suppressive response by acephate depict a complex pattern of CYP modulation with the kidney being more responsive to 3A induction (up to 6.9-fold increase, male) and the lung to 2B1 suppression (up to 70% loss, mainly female). In the liver, a 2.7-fold increase in the 3A-like activity, probed by the O-demethylation of aminopyrine, in the O-deethylation of phenacetin (1.8-fold increase, 1A2), as well as in the hydroxylation of p-nitrophenol (1.6-fold increase, 2E1) was observed in male animals at a lower dose. In contrast, a marked reduction of CYP 1A1-mediated ethoxyresorfin O-deethylase activity ranging from 43% (lower dose) to 44% loss (higher dose) in female and male mice, respectively, and of CYP 2B1-mediated pentoxyresorufin O-dealkylase (3% loss, female) was achieved. In the kidney, an increase in the 'mixed' ethoxycoumarin O-deethylase (up to 2-fold) as well as in the 2B1-like activity (up to 2.8-fold) was also recorded in males at 250 mg/kg. Once again, in the lung, a different behaviour on 3A isoforms between female (approximately 2-fold increase) and male (44% loss) was seen at a lower dose. The specificity of CYP changes was corroborated by means of Western immunoblotting analysis using rabbit polyclonal antibodies, anti-CYP 3A1/2 and 2E1. Taken together, these data indicate a possible toxic/cotoxic, cocarcinogenic and promoting potential of acephate.

A database for evaluating the toxicological risk of pesticides.

This study of Overtox-DB, a computerized database for managing chemical toxicity data, is a product of the application of typical methodologies regarding information science and computer technology. The methodology applied can be reduced to three-basic elements: the collection of requirements, design, and achievement. Overtox-DB was developed by defining technological elements for managing data and its structure and by identifing the procedures and methodologies for data storage, retrieval, distribution, and standardization of many kinds of test data stored in the same format. The program stores data about chemical identification, physical and chemical properties, toxicological tests, mutagenicity, teratogenicity, carcinogenicity, and a bibliography of chemical compounds. Overtox-DB consists of five modules: experimental and bibliographic, data collection, molecular data collection, data search, and data report. The Overtox-DB user responds to a simplified set of query commands and boolean operators that interact with the system to retrieve different toxicological data (the majority of fields are defined as search fields and identify the test system, results of the assays, administration route, dose, etc.). The collected information provides an analytical characterization of biological activities for many compounds and identifies evidence possibly lacking in experimental approaches. Indeed, this database could permit a comparative evaluation with other substances and can be used for structure-activity relationship studies.

Cytotoxic and cell transforming activities of the fungicide methyl thiophanate on BALB/c 3T3 cells in vitro.

Cytotoxic and cell-transforming activities of methyl thiophanate a systemic fungicide capable of entering plant cells and thus controlling fungal diseases that have already started were studied in an in vitro medium-term (6-8 weeks) experimental model utilizing BALB/c 3T3 cells. Cells were exposed to the chemical, dissolved in dimethyl sulfoxide, in the absence or presence of an exogenous metabolizing system derived from rat livers supplemented with cofactors (S9 mix). In the absence of metabolic activation, methyl thiophanate exerted cytotoxic activity, evidenced through the formation of cell colonies, at low doses (> 10 micrograms/ml). However, the cytotoxic activity was greatly reduced by the S9 mix-induced metabolic activation of the chemical. Without bioactivation, cell-transforming potential, evidenced through the induction of transformation foci, was observable only at the highest (weakly toxic) dose employed (25 micrograms/ml). On the contrary, in the presence of metabolic activation, the cell-transforming activity was detectable at all tested doses (i.e. from 20 to 200 micrograms/ml) and it was particularly evident in a level-II transformation amplification test when the cells were allowed to perform active proliferative activity. These results, providing further information on the activity of methyl thiophanate in multistep carcinogenesis as possible genotoxic and/or co-carcinogenic agent, may contribute to better evaluate the oncogenic risk to man.

Cytotoxic activity and transformation of BALB/c 3T3 cells in vitro by the insecticide acephate.

Cytotoxic and cell transforming activity of the organophosphate insecticide acephate have been studied in an in vitro experimental model which foresees the exposure of BALB/c 3T3 cells to the chemical. The assay was performed in the presence or absence of metabolic activation system derived from phenobarbital and beta-naphthoflavone induced rats (S9-mix). Cytotoxicity of acephate was unaffected by the presence of the metabolizing fraction. Cell-transforming potential, evidenced through the induction of transformation foci, was observed at all tested doses (i.e. 100, 200 and 400 micrograms/ml) with or without exogenous bioactivation. This activity was related with cell proliferation since it was particularly evident in a level-II cell-transformation assay when the cells were allowed to perform active proliferative activity. These findings, obtained in a medium-term (6-8 weeks) test, may contribute to a better understanding of the action of acephate in the multistep carcinogenesis, proving more information on the oncogenic risk to humans.

Enhancement of BALB/c 3T3 cells transformation by 1,2-dibromoethane promoting effect.

Two of the most representative halogenated aliphatic hydrocarbons, 1,2-dibromoethane and 1,1,2,2-tetrachloroethane, were tested in the two-stage cell transformation model for analysing the promoting ability. Both of these compounds had previously been found to exert genotoxic effects, probably acting as moderate initiators. BALB/c 3T3 cells were initiated with subtransforming doses of N-methyl-N-nitro-N-nitrosoguanidine or 3-methylcholanthrene and then exposed to a chronic treatment with different non-transforming dosages of the two haloalkanes. 1,1,2,2-Tetrachloroethane did not exert any promoting activity in that system. By contrast, significant promoting effects by 1,2-dibromoethane were observed both in cells treated with N-methyl-N-nitro-N-nitrosoguanidine and in cells treated with 3-methylcholanthrene. Promotion of the transformation process initiated with 3-methylcholanthrene was detectable when confluent cells in the chemical-treated plates were replated in the level-II amplification test. This experimental procedure allowed cells to perform further rounds of replications and transformed foci to became detectable. Results gave evidence for a promoting role of 1,2-dibromoethane in multistep carcinogenesis, probably responsible for the higher oncogenic ability of this compound with respect to 1,1,2,2-tetrachloroethane.

Transformation of BALB/c 3T3 cells in vitro by the fungicides captan, captafol and folpet.

Cytotoxic and cell-transforming activities of the three fungicides, captan, captafol and folpet, have been studied in an experimental in vitro model by exposing BALB/c 3T3 cells to the chemicals with or without S-9 mix-induced bioactivation. Cytotoxicity of the three compounds was reduced in the presence of the metabolizing system. Each assayed pesticide displayed cell-transforming ability in the presence of the metabolizing system. The relative efficiency was: captafol > captan > folpet. Cell transformation was considered to be due to carcinogenesis-promoting activity. These data, obtained in a medium-term (6-8 weeks) experimental model, contribute to a better understanding of the action of the three pesticides in the multistep carcinogenesis process and provide more information concerning the oncogenic risk of these xenobiotic compounds for humans.

Cytotoxic and cell transforming effects of the insecticide, lindane (gamma-hexachlorocyclohexane) on BALB/c 3T3 cells.

Further information was gathered on the possible carcinogenic hazard associated to the exposure to the insecticide lindane (gamma-hexachlorocyclohexane). The parameters studied were the cytotoxic and cell transforming activities of the pesticide on BALB/c 3T3 cells in an in vitro experimental model system in the absence or in the presence of rat liver S-9 mix-induced metabolic activation of the chemical. Lindane did not exert cytotoxic effects at all the tested doses (ranging from 10 micrograms/ml to 200 micrograms/ml) in the absence of bioactivation. However, dose-related cytotoxic effects were observed in the presence of the metabolizing system. Furthermore, lindane showed statistically significant and dose-dependent cell transformation activity at all the tested doses (10 micrograms/ml, 50 micrograms/ml and 100 micrograms/ml ) either in the absence or in the presence of bioactivation. This activity was related with cell proliferation since it was exerted in a level-II transformation test by replating cells and allowing the amplification of the cell transforming effects of the chemical. The formation of radicals and of reactive oxygen species, resulting from the chemical metabolism, could account for lindane activity as carcinogenesis promoting agent, although contemporary genotoxic effects induced by the pesticide could not be excluded.

1,2-Dibromoethane as an initiating agent for cell transformation.

The two-stage transformation assay increases the sensitivity of cells to chemicals and permits detection of carcinogens acting as initiating agents. 1,2-Dibromoethane, a representative halogenated aliphatic, has been tested in the two-stage BALB/c 3T3 cells transformation test at dosage from 16 microM to 128 microM. This dose range is much lower than those previously found efficient in transforming BALB/c 3T3 cells. Apart from the lowest dose, which induced borderline effects, all the other assayed dosages appeared to induce heritable changes in the target cells. The initiated cells were revealed as fully transformed foci both in the combination with a chronic promoting treatment and also by allowing cells to perform more rounds of cell replication. The results clearly show that 1,2-dibromoethane can act as an initiator of cell transformation.

In vitro transforming effect of the fungicides metalaxyl and zineb.

The cytotoxic effects and the transforming properties of two fungicides, metalaxyl and zineb, whose mutagenic or carcinogenic activity has not been clarified yet, were analyzed in the in vitro BALB/c 3T3 cell transformation test both in the presence and in the absence of an exogenous metabolizing system. Zineb was completely detoxified when the exogenous metabolizing system was added to the target cells to increase their inherent metabolic capacity. Metalaxyl induced cell transformation at any assayed dosage, i.e., 500, 250, and 50 micrograms/ml, in the presence of bioactivation, and at the highest dosage (500 micrograms/ml) in the absence of bioactivation. The transforming effect was detectable only in the level-II transformation cultures and it was likely linked to the induction of additional cell proliferation which allowed obtaining the transformation amplification in these experimental conditions.

Genetic safety evaluation of pesticides in different short-term tests.

Cyanazine, cyhexatin, dicamba and DNOC are pesticides commonly and broadly used in agriculture pest control. However, there is little information on their toxicity and mutagenicity in human cells and in whole animals. Therefore, UDS assay and SCE assay in human peripheral lymphocytes, and chromosome aberration analysis in bone marrow of rats have been used to assess the DNA-damaging activity of the above pesticides. Cyanazine proved non-genotoxic in all the test systems. Cyhexatin showed only weakly positive results for SCE induction in human lymphocytes, providing no concern for genotoxicological hazard. While dicamba did not show clastogenic effects in rodents, DNOC gave significant dose-related increases of structural chromosome aberrations in rat bone marrow cells. Female animals showed increased sensitivity to the toxic effects by DNOC at the highest dose. The results provide further information on the intrinsic genotoxic activity of the tested pesticides, which may contribute to the toxicological assessment of the risk associated with human exposure.

In vitro cell transformation induced by the pesticide fenarimol.

The pesticide fenarimol is capable of transforming BALB/c 3T3 cells in an in vitro model system, and its action resembles a carcinogenic process in vivo. In the absence of metabolic activation, transformed foci are already visible in the standard experimental procedure. The addition of the S9 fraction as an exogenous metabolic system leads to a decrement of cytotoxic effects and the reduction of the transformation rate. The transformed phenotype, however, becomes visible when confluent cells are replated and allowed further cell replication. Transformation effects by fenarimol may be due to both a weak genotoxic activity and/or stronger promoting activity.