Mutagenic and toxic effects of 4-hydroperoxycyclophosphamide and of 2,4-tetrahydrocyclohexylamine (ASTA-Z-7557) on human lymphocytes cultured in vitro.

Certain biological effects exerted by 4-hydroperoxycyclophosphamide and by 2,4-tetrahydrocyclohexylamine (ASTA-Z-7557), utilized in vitro in the therapy of leukemias and lymphomas to eliminate the occult tumor cells in autologous marrow transplantations, were studied in human lymphocytes cultured in vitro. The data show that these drugs exert mutagenic activity eliciting unscheduled DNA synthesis (reparative synthesis) after DNA damage and cause about tenfold higher frequency of sister chromatid exchanges than controls. Furthermore, they exert strong toxic effects, measured as tritiated thymidine uptake inhibition, on mitogen-stimulated dividing cells even if pretreated during the nonproliferative phase of the cell cycle in which the toxic activity of the drugs is not detectable. Data obtained with doses of the drugs similar to those used in the therapy are discussed in terms of the therapeutic use of these chemicals.

DNA damage by haloalkanes in human lymphocytes cultured in vitro.

The DNA damaging activity of 7 haloalkanes was studied in a short-term in vitro system which utilized human lymphocytes. The parameters studied were the inhibition of scheduled (duplicative) and unscheduled (reparative) DNA syntheses seen as tritiated thymidine uptake. The results obtained suggested that chloromethyl methyl ether (CMME), 1,2-dibromoethane (DBE), trichloroethylene (TCE) and 1,2-dichloroethane (DCE) gave positive results such as DNA damaging agents, while carbon tetrachloride (CTC), chloroform (TCM) and dichloromethane (DCM) gave low or negative results.

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.

Initiating activity of 1,1,2,2-tetrachloroethane in two-stage BALB/c 3T3 cell transformation.

By using in vitro two-stage BALB/c 3T3 cell transformation assay, we have tested the effect of promoting treatment with tetradecanoylphorbol acetate (TPA) on transformation induced by 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE). Cells were treated with subeffective or transforming concentrations of 1,1,2,2-TTCE in the presence of an S9-mix activating system, followed by TPA promoting treatment. The transforming activity of 1,1,2,2-TTCE is evident only by reseeding confluent cells and allowing additional rounds of cell replications in the amplification test. Treatment with TPA leads to a marked transformation yield in all plates scored even at the lowest assayed dosage of 1,1,2,2-TTCE, without performing amplification of transformation.

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.

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.

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.

GM-CSF incubation prior to treatment with cytarabine or doxorubicin enhances drug activity against AML cells in vitro: a model for leukemia chemotherapy.

We studied the effect of preincubation with recombinant GM-CSF on the activity of cytarabine and doxorubicin against clonogenic acute myeloid leukemia cells (CFU-AML). Leukemia cells from seven persons with AML, three myeloid cell lines (HL60, KG1, K562) and two control cell lines (U937, MOLT3) were tested. Preincubation with GM-CSF (0.01-0.1 microgram/ml) increased DNA synthesis as measured by tritiated thymidine incorporation and intranuclear Ki67 expression in cells from six persons with AML and in HL60 cells. Leukemia cells preincubated with GM-CSF for 6-48 h were exposed to cytarabine (2-200 micrograms/ml) or doxorubicin (0.01-0.1 microgram/ml) for 3 h and CFU-AML assayed. This approach further reduced CFU-AML in samples from six persons with AML and in HL60 and KG1 cells compared to cells not preincubated with GM-CSF prior to drug treatment. In most instances, reduced CFU-AML correlated with GM-CSF induced DNA synthesis. These data suggest a possible strategy of GM-CSF pretreatment to increase anti-leukemia efficacy of chemotherapy in AML.

In vitro cytotoxic and cell transforming activities exerted by the pesticides cyanazine, dithianon, diflubenzuron, procymidone, and vinclozolin on BALB/c 3T3 cells.

Cytotoxic and cell transforming activities of the pesticides cyanazine, diflubenzuron, dithianon, procymidone, and vinclozolin were investigated in vitro by utilizing the BALB/c 3T3 cell transformation test performed in the presence or in the absence of S-9 mix as an exogenous bioactivation system for the chemicals. All the assayed pesticides were cytotoxic in the absence of S-9 mix, whereas only dithianon exerted cytotoxic effects in the presence of metabolic activation. All the chemicals tested did induce BALB/c 3T3 cell transformation, to a various extent, in the absence of S-9 mix. Cell transforming ability of cyanazine and diflubenzuron was not detectable in the presence of S-9.

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.

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.

Purification, characterization and biological activity of tulipin, a novel inhibitor of DNA synthesis of plant origin.

A DNA synthesis-inhibiting protein (for which the term tulipin is proposed) was isolated from the bulbs of Tulipa sp. The yield ranged from 3.4 to 4.1 per cent of total protein content of the crude extract. Mr, isoelectric point, neutral and amino sugar and amino acid composition were determined. Inhibition of DNA synthesis varied in intact cells according to the cellular types studied, with a minimum ID 50% (concentration giving 50% inhibition) of 400 ng/ml in neuroblastoma cells. The effect was reversible. No effect was obtained in cell-lysate. RNA and protein synthesis were unaffected. The acute toxicity, evaluated in Swiss mice, gave an LD of 6.1 mg/kg body wt. Results of electron microscopy are also given. A second protein, called tulipin 2, has been isolated and partially characterized.

Toxic activity of seventeen industrial solvents and halogenated compounds on human lymphocytes cultured in vitro.

Seventeen chemicals (solvents, insecticides and intermediates in the production of textiles and resins) were tested in a short-term in vitro system with human lymphocytes to determine their toxic action. The parameters studied were the tritiated thymidine uptake and cell viability in cultures grown with or without a rat liver metabolizing system (S-9 mix). Data obtained showed that 1,3-dichlorobenzene, 1,2-dichlorobenzene, hexane, 1,2-diiodoethane, 1,4-dichlorobenzene, tetrachloroethylene, 2,3-dibromopropanol, chloromethyl methyl ether, 1,2- and 1,3-dibromopropane, in order, exerted the more toxic effects; ethyl acetate, cyclohexane, cyclohexanone and benzene showed lower toxic activity. The chemicals lost their toxic power in the presence of the metabolizing system with the exception of 1,2- and 1,3-dichlorobenzene which maintained in some degree their toxicity even in the presence of the S-9 mix. Only chloromethyl methyl ether elicited unscheduled DNA synthesis acting as DNA damaging agent.

The covalent interaction of 1,4-dibromobenzene with rat and mouse nucleic acids: in vivo and in vitro studies.

1,4-Dibromobenzene (1,4-DBB) was covalently bound to DNA from liver, kidney, lung and stomach of mice after intraperitoneal administration. The covalent binding index (CBI) value (23 in mouse liver) was typical of weak initiators. On the contrary, no interaction with DNA from rat organs was observed (CBI detection limit: 1.3-2.6). The in vitro interaction of 1,4-DBB with calf thymus DNA was mediated mainly by microsomes, especially those from liver of both species and from mouse lung. Mouse subcellular fractions were more active then rat subcellular fractions. Unlike liver cytosol, subcellular cytosolic fractions from lung, kidney and stomach were capable of bioactivating 1,4-DBB, although to a lesser extent than liver microsomes. Both cytochrome P-450 and GSH-transferases are involved in 1,4-DBB bioactivation.

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.

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.

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.

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.

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.

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.