Cancer chemoprevention: some complications and limitations.

Chemopreventive strategies are very attractive and have earned serious consideration as a potential means of controlling cancer incidence. However, the use of some anti-initiating entities (enzyme inducers or inhibitors) devised to reduce tumor initiation is controversial. Indeed, considering the double-edged-sword (activating or detoxifying) nature of drug metabolizing enzymes, any attempt to modulate such catalysts by dietary components (including drugs) may lead to cancer risk.

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.

Biomarkers of effect in evaluating dithianon cocarcinogenesis: selective induction and suppression of murine CYP3A isoform.

The ability of dithianon, whose mutagenic/cocarcinogenic activity has as yet not been clarified, to affect specific biomarkers of effect related to non-genotoxic cocarcinogenesis was investigated. For this purpose, several CYP-dependent reactions have been studied in liver, kidney and lung microsomes derived from male and female Swiss Albino CD1 mice treated i.p. with single (3 or 6 mg kg(-1) b.w.) or repeated (3 mg kg(-1) b.w., daily for 3 days) administrations of such fungicide. No significant changes in both absolute and relative liver, kidney and lung weights were achieved after dithianon treatment. Whereas a single dose was able to significantly induce certain monooxygenases, with repeated treatments a loss of activity was observed. For example, a approximately 2.4-fold increase of CYP3A-dependent activity, probed by N-demethylation of aminopyrine, was achieved in the liver (both sexes, lower dose) and, to a lesser extent, in lung. A small, but significant increase in the hydroxylation of p-nitrophenol (2E1) and in the O-deethylation of ethoxycoumarin (mixed) was also found in liver. With the exception of a approximately 46% loss in the 3A-like activity, no appreciable changes of the selected biomarkers were observed in kidney. Repeated dithianon doses were able to significantly reduce the 3A- and 2E1-dependent monooxygenases (approximately 30% and approximately 30% loss, respectively, averaged between male and female), as well as ethoxycoumarin O-deethylase activity (approximately 54% loss) in the liver. On the contrary, no significant CYP modulation in both kidney and lung was recorded. On the whole, dithianon has a complex pattern of CYP induction or suppression in various tissues of both sexes, suggesting the possible toxic/cotoxic and cocarcinogenic potential of this fungicide. These data can contribute to a better understanding of its toxicological profile, providing more information concerning the risk associated to human exposure.

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.

Biomarkers of effect in evaluating metalaxyl cocarcinogenesis. Selective induction of murine CYP 3A isoform.

The ability of metalaxyl, whose mutagenic/cocarcinogenic activity has as yet not been clarified, to affect specific biomarkers related to non-genotoxic cocarcinogenesis, was investigated. Several CYP-dependent reactions have been studied in liver, kidney and lung microsomes derived from male and female Swiss Albino CD1 mice treated i.p. with single (200 or 400 mg/kg b.w.) or repeated (200 mg/kg b.w., 3 days) administrations of fungicide. No significant changes in both absolute and relative liver, kidney and lung weights were observed after metalaxyl treatment. Although a single dose did not significantly affect the considered monooxygenases, a clear example of selective CYP3A induction was recorded in different tissues after repeated treatment. A 3 approximately -fold increase in CYP3A isozymes, probed by N-demethylation of aminopyrine, was observed in the liver (both sexes). Again, a 5 approximately -fold increase (averaged between male and female) in this oxidase activity was present in the kidney. No significant change of the selected biomarkers was observed in the lung. A weak, but significant reduction of CYP2B1 isoform in liver (male) was also recorded. Liver and kidney CYP3A overexpression was corroborated by means of Western immunoblotting analysis using rabbit polyclonal antibodies anti-CYP3A1/2. Northern blotting analysis with CYP3A cDNA biotinylated probe showed that, in the liver, the expression of this isozyme is regulated at the mRNA level. On the whole, these data seem to indicate the cotoxic and cocarcinogenic potential of this fungicide.

Paramagnetic resonance in detecting carcinogenic risk from cytochrome P450 overexpression.

BACKGROUND: Despite the increasing interest in the role of oxygen radicals on human degenerative disorders including cancer, oxidative stress status is not yet measurable in vivo, largely precluding clinical application. Limited semi-quantitative assays of damage to broad classes of biomolecules such as lipids, proteins, and DNA are currently available. The detection of radicals in humans by a whole-body electron paramagnetic resonance (EPR) technique has not yet been developed, although this possibility has long fascinated free radical investigators. METHODS: While the EPR spin trapping procedure can be used to detect carbon centered or hydroxyl radical in human tissues, the most common spin traps are much less useful for capturing the superoxide anion (O2). To overcome these limitations, we propose a whole-body-harvest approach that utilizes a highly lipophilic spin scavenger that when injected in the animal is capable of trapping the O2 generated in vivo throughout the body with formation of a stable nitroxide measurable by EPR in the urine. A process known to generate the O2 is the induction of certain cytochrome P450 (CYP) isozymes by drugs or environmental pollutants. RESULTS: We report: 1) a correlation between the induction of each CYP gene family and the O2 yield; 2) support to an observation reported previously that the tumor promoting ability of CYP inducers is mainly mediated by the O2; and 3) the description of a method for nitroxide mediated O2 detection in vivo. CONCLUSION: These findings could open the way for using electron spin resonance in diagnostic practice.

The genetic and non-genetic toxicity of the fungicide Vinclozolin.

The mutagenic/cocarcinogenic potential of the fungicide Vinclozolin was assessed by a comprehensive examination of toxicity mechanisms at both the genetic and the metabolic level. Vinclozolin did not induce any significant increase in chromosomal aberrations in human pheripheral blood lymphocytes cultured in vitro, both in the presence and in the absence of metabolic activation. However, significant dose-related increases in micronucleated erythrocytes (up to 4-fold over the control) were found in the bone marrow cells of mice 24 h after treatment with the fungicide over a range of concentrations from 312.5 to 1250 mg/kg. The morphology and the size of micronuclei induced was suggestive of a predominantly clastogenic mode of action. Several cytochrome P450 (CYP)-dependent reactions have been monitored in liver, kidney and lung microsomes of male and female Swiss Albino CD1 mice in order to ascertain certain toxic non-genetic properties (related to carcinogenesis) of Vinclozolin. It was found to be a selective inducer towards CYP 3A (liver, kidney) and 2E1 (liver), as exemplified by the significant increases of the demethylation of aminopyrine (APND, up to 2.3-fold, female liver), and hydroxylation of p-nitrophenol (pNPH, up to 5.6-fold, male liver). In general, however, Vinclozolin has a complex pattern of induction and suppression of CYP-dependent enzymes, as shown from the reduced expression of various monooxygenases depending upon dose, sex or organ considered. For example, pNPH activity was suppressed in kidney (up to 48% loss, averaged between male and female), whereas ethoxycoumarin O-deethylase was reduced in lung up to 53% in male (at the highest dose). These data were sustained by means of Western immunoblotting using rabbit polyclonal antibodies anti-CYP 3A and 2E1. Northern blotting analysis using CYP 3A1/2 and 2E1 cDNA biotinylated probes showed that the expression of such isozymes is regulated at the mRNA level. Taken together, the findings indicate the clastogenic activity and the possible cotoxic, cocarcinogenic and promoting potential of Vinclozolin.

Genetic and non-genetic biomarkers related to carcinogenesis in evaluating toxicological risk from Fenarimol.

A multibiomarker approach based on the study of toxicity mechanisms at both genetic and metabolic levels has been applied to Fenarimol. With regard to genotoxicity, particular attention was given to assays for chromosomal aberration and micronuclei; clastogenic potential was assessed in human peripheral blood lymphocytes in vitro, while the induction of micronuclei was studied in male CD1 mouse bone marrow polychromatic erythrocytes (PCE). Fenarimol did not induce any significant dose-related increase in micronucleated PCEs, up to 4-fold above the control level at a single dose of 75 mg/kg b.w., was observed 24 h after treatment. Using selective biochemical markers of effect Fenarimol was found to induce CYP 2B1 isoforms in liver, kidney and lung microsomes of Swiss Albino CD1 male and female mice, as shown by the significant increase in specific 2B1-probe pentoxyresorufin O-dealkylase activity. On the contrary, CYP 3A, probed by N-demethylation of aminopyrine, were only induced in the liver. Results were corroborated by means of Western immunoblotting using rabbit polyclonal antibodies anti-CYP 2B1 and 3A. Northern blotting analysis with CYP 2B1 and 3A cDNA biotinylated probes showed that the expression of such isoforms is regulated at mRNA level. Taken as a whole, these data indicate the possible (mutagenic) cotoxic/cocarcinogenic and promoting potential of this fungicide.

Molecular non-genetic biomarkers related to Fenarimol cocarcinogenesis: organ- and sex-specific CYP induction in rat.

Selective biochemical markers of effect have been used to evaluate some non-genotoxic cocarcinogenic properties of Fenarimol. Several CYP-dependent reactions have been monitored in liver, kidney and lung microsomes of male and female Sprague-Dawely rats treated (i.p.) with 200 or 400 mg/kg body wt dose of this pesticide. Highly specific substrates were used as probes of various isoforms, such as CYP1A1, 1A2, 2B1, 2E1 and 3A. A complex pattern of CYP induction, including organ- and sex-related differences in the inductive response by Fenarimol, has been recorded in this investigation, the kidney (mainly male) being more responsive when compared to other tissues. A 6.6-fold increase in the 2B1-like activity, probed by dealkylation of pentoxyresorufin was observed in the liver at a higher dose. On the contrary, a marked induction of CYP1A1 mediated ethoxyresorufin O-deethylase activity, ranging from 20- to 35-fold in female and male, respectively, was observed in the kidney at a lower dose tested. In the lung, at a higher dose, the p-nitrophenol hydroxylase activity (2E1) was enhanced up to 3.5-fold in male animals, whereas the 3A-like activity, probed by the N-demethylation of aminopyrine, was induced up to 2.6-fold in females. A weak, although significant reduction of CYP2B1 isoforms in lung was also recorded. Taken together, these data corroborated by means of Western immunoblotting analysis (using rabbit polyclonal antibodies anti-CYP 2B1/2, 1A1, 2E1, and 3A1/2) indicate a possible cotoxic, comutagenic cocancerogenic and promoting potential of this fungicide.

Testosterone hydroxylase in evaluating induction and suppression of murine CYP isoenzymes by fenarimol.

This study aimed to investigate the effect of single and repeated administration of fenarimol on murine liver, kidney and lung microsomal CYP-catalyzed drug metabolism. The modulation of the regio- and stereo-selective hydroxylation of testosterone by fenarimol was considered in evaluating cocarcinogenic properties. Induction or suppression of different CYPs was recorded after a single dose of the fungicide. For example, in liver, 6 beta-(mainly associated with CYP3A), 7 alpha- and 2 beta-testosterone hydroxylase (TH) activities were induced up to 4.8-fold (7 alpha-TH) in female mice, at a dose of 150 mg/kg. In contrast, at 150 and 300 mg/kg, 16 alpha-TH (CYP2B9), 17-TH (female) and 6 alpha-TH (CYP2A1 and 2B1, male) activities were appreciably reduced. In extrahepatic tissues, the CYP modulation pattern was different, 16 alpha-TH being the only metabolite decreased (lung, male). In kidney, 16 beta-TH and 17-TH activities were increased up to 5.8-fold in female mice (lowest dose), while in lung 6 alpha-TH and 7 alpha-TH activities were induced up to 6- and 7-fold, respectively (both doses). Repeated treatment (150 mg/kg for 3 days) was able markedly to induce all steroid hydroxylations, up to 78-fold in 2 alpha-TH activity (male liver). In conclusion, fenarimol has a complex pattern of CYP induction or suppression in various tissues of both sexes, suggesting the possible toxic, cotoxic/cocarcinogenic and promoting potential of this fungicide.

Induction of CYP2B1 mediated pentoxyresorufin O-dealkylase activity in different species, sex and tissue by prototype 2B1-inducers.

The induction of CYP2B1 mediated pentoxyresorufin O-dealkylase (PROD) activity by various xenobiotics was explored in liver, kidney and lung from a variety of animal species of both sexes, in order to gain insights into the substrate specificity of induced CYPs. Marked species- and sex-related differences in the inducibility of PROD activity by tested chemicals were observed, the mouse being always more responsive when compared to hamster or rat. Induction by sodium phenobarbital (NaPB) led to a conspicuous increase in all situations, up to approximately 38-fold in female rat and mouse liver, with the exception of hamster kidney where PROD activity was only slightly affected. Unexpectedly, both sodium barbital (NaB) and phorone (PHR) moderately induce CYP2B1 isoforms in rat, the extent being highest in female kidney (PHR, 14-fold increase) and male lung (NaB, 4.5-fold). The degree of induction was maximal in the liver with some exceptions occurring in male mice where NaB induced up to 46- and 115-fold increases in lung and kidney and PHR up to 115-fold in kidney. Minimal, although significant induction of PROD activity following treatment with trans-1,2-dichloroethylene (1,2-DCE) occurred in all situations with the exception of hamster kidney and lung. Therefore, caution should be exercised when using PROD activity as specific enzymatic assay to probe CYP2B1-like induction.

The modulating activity of interferon on benzo(a)pyrene bioactivation and clastogenesis in mice.

Acute intraperitoneal administration of benzo(a)pyrene (80 mg/kg b.wt.) resulted in time-dependent increases in chromosome aberrations, especially of break-type in the bone marrow of treated mice. Pretreatment with murine interferon-alpha/beta (5 x 10(4) IU daily for two days) caused a significative decrease in the cytogenetic response in vivo of benzo(a)pyrene (up to 51%) and a stabilization of aberrant cells up to 48 hr. The administration of murine interferon-alpha/beta gave rise to a marked depression of microsomal monooxygenase system after 24 hr, as exemplified by the significant reduction of cytochrome P450 content as well as deethylation of ethoxyresorufin. Interferon treatment delayed the obtainment of basal levels of oxidative metabolism to approximately 30 hr. After interferon plus benzo(a)pyrene treatment, ethoxyresorufin O-deethylase activity showed a reduction up to 60%; levels comparable to benzo(a)pyrene treated group were restored by 48 hr. Immunoblotting analysis confirmed reduced CYP1A1 level. Results suggest that the inhibition of benzo(a)pyrene hepatic metabolism by interferon was reflected by changes in its clastogenic activity. Persistence of low level of chromosome aberration at 48 hr may be reconducible to other interferon sensitive processes than effects on hepatic mixed-function oxidase system, such as DNA repair activity and cell proliferation.

Strategies for advancement of short-term mutagenicity tests: on the optimal ionic strength for the liver microsomal assay.

The aim of this work was to optimize the ionic strength (tau) in the liver microsomal assay (LMA) in performing short-term genotoxicity tests. tau optimization would increase the sensitivity (i.e. decrease false negatives) and at the same time increase the specificity (decrease false positives). Such optimization depends upon the relative activities and stabilities of the liver polysubstrate cytochrome P450- and FAD-containing monooxygenase-dependent metabolizing enzymes present in the incubation mixtures. With regard to phase-I pathway, the expression of various P450-like activities (IA1, IA2, IIB1, IIE1, IIIA P450 classes) and thiobenzamide s-oxidase (as FAD-MFO marker), were examined in terms of their exact incubation conditions for the LMA during a period of preincubation (1 h) over the tau range 0.06-1.40. As a comparison with the phase-II pathway, the behaviour of glutathione S-transferases (total and pi class), glutathione S-epoxide transferase, epoxide hydrolase and UDP-glucuronosyl transferase were studied. Lipid peroxidation (LP) was also determined. Experiments were performed on S9 fractions derived from sodium phenobarbital, beta-naphthoflavone, isosafrol, ethanol and pregnenolone 16-alpha carbonitrile super-induced mouse liver. The maximal value of the mean specific activity (Asp), up to a 46% increase, was found at tau = 0.864 for oxidative reactions considered. On the contrary, a slight modulation of Asp for post-oxidative reactions was seen. LP was not changed appreciably by varying tau. In vitro DNA binding of the well-known premutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at tau = 0.864 (2.25-fold) compared to the usual tau (0.06) used. Additional confirmation of these results stems from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. Indeed, a significant enhancement of mitotic gene conversion (up to 1.8-fold), mitotic crossing-over (2.6-fold) and reverse point mutation (2.6-fold) frequencies was achieved at tau = 0.86 compared to tau = 0.06 (traditional). These data show that tau = 0.86 can provide more convenient conditions for in vitro bioactivation (as exemplified by an increased Asp phase-I/Asp phase-II ratio), as well as DNA binding and genotoxic response.

On the hepatotoxicity of 1,1,2,2-tetrachloroethane.

Intoxication of male and female mice with a single dose (300 or 600 mg/kg) of 1,1,2,2-tetrachloroethane (TTCE) resulted in significant decreases in cytochrome P-450 (to 58-73% of the control) and NADPH-cytochrome (P-450) c-reductase (to 29-35% of the control) in hepatic microsomes. This was accompanied by an alteration of mixed function monooxygenases stemming from the marked reduction (to 20-64% of the control) of several oxidative activities to selected substrates towards different P-450 isozymes (classes IA1, IA2, IIB1, IIE1 and IIIA). As phase II markers, epoxide hydrolase (approximately 35% loss), UDP-glucuronosyl transferase (approximately 42% loss) and to a lesser extent glutathione S-transferase (approximately 17% loss) were all affected. Also, the activity of delta-aminolevulinic (ALA) synthetase was decreased (approximately 57% of the control). On the contrary, heme oxygenase activity was increased (up to 35%) at the maximal dose tested. The decrease of P-450-function may be explained in terms of an alteration in the rate of heme biosynthesis and degradation, provoking a loss of heme content (approximately 33%) as well as of the direct inactivation of both P-450 and reductase. Because of increasing evidence on the involvement of free radical intermediates in the case of toxicity of haloalkanes, electron spin resonance spectroscopy (ESR) spin-trapping in vivo techniques were used to characterize the possible free radical species involved in the observed liver damage. The results obtained with the spin-trap N-benzylidene-2-methylpropylamine N-oxide (phenyl t-butylnitrone, PBN) provide evidence for the formation and trapping of the CHCl2CHCl free radicals. The detection of conjugated diene signals by means of second-derivative spectrophotometry, have enabled us to show that in vivo lipid peroxidation may be one of the main mechanisms responsible for TTCE hepatotoxicity.