R. Mesirca

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.

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.

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

Abstract 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, 6β-(mainly associated with CYP3A), 7α- and 2β-testosterone hydroxylase (TH) activities were induced up to 4.8-fold (7α-TH) in female mice, at a dose of 150 mg/kg. In contrast, at 150 and 300 mg/kg, 16α-TH (CYP2B9), 17-TH (female) and 6α-TH (CYP2A1 and 2B1, male) activities were appreciably reduced. In extrahepatic tissues, the CYP modulation pattern was different, 16α-TH being the only metabolite decreased (lung, male). In kidney, 16β-TH and 17-TH activities were increased up to 5.8-fold in female mice (lowest dose), while in lung 6α-TH and 7α-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 2α-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.

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.

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.

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.

Selective induction of murine liver cytochrome p450 IIB1 by Halogenated hydrocarbons

Thirteen halogenated hydrocarbons, 1,1,1,2‐tetrachloroethane (1,1,1,2‐TCE), 1,1,2,2‐tetrachloroethane (1,1,2,2‐TCE), pentachloroethane (PCE), 1,2‐dibromoethane (DBE), 1,1,2,2‐tetrabromoethane (1,1,2,2‐TBE), iodoethane (IE), 1,1‐dichloroethane (1,1‐DCE), 1,1,1‐trichloroethane (1,1,1‐TCE), 1,1,2‐trichloro‐ethane (1,1,2‐TCE), dichloromethane (DCM), 1,1‐dichloroethylene (1,1‐DCEE) and trans 1,2‐dichloro‐ethylene (t1,2‐DCE) were evaluated for their ability to modulate specific cytochrome P450 (cyt P450) isoforms following in vivo administration in male mice. Each chemical was administered (i.p.) in equivalent doses (50, 25, 12.5 and 6.25% of the respective LD50). Total cyt P450 and selected microsomal monoxygenase activities towards different P450 isoenzymes (classes IA1, UBI, IIE1 and IIIA P450) were examined in hepatic microsomes. With the exception of 1,1‐DCE, all considered hydrocarbons were inducers of Cyp2B1 genes, as exemplified by the enhancement of pentoxyresorufin O‐dealkylation. A 1.5‐(DBE, DCM) to ...

Correlation between murine liver cytochrome p450 2b1 induction by halogenated hydrocarbons and toxicity

The aim of this investigation was to study the relationship between the induction of cytochrome P450 2B1 isozymes by thirteen halogenated hydrocarbons and their toxicity. Each chemical, 1,1,2,2‐tetrabromoethane (1,1,2,2‐TBE), 1,1,1,2‐tetrachloroethane (1,1,1,2‐TCE), 1,1,2,2,‐tetrachloroethane (1,1,2,2‐TCE), pentachloroethane (PCE), 1,2‐dibromoethane (1,2‐DBE), iodoethane (IE), 1, 1‐dichloroethane (1,1‐DCE), 1,2‐dichloroethane (1,2‐DCE), 1,1,1‐trichloroethane (1,1,1‐TCE), 1,1,2‐trichloroethane (1,1,2‐TCE), dichloromethane (DCM), 1,1‐dichloroethylene (1,1‐DCEE) and trans 1,2‐dichloroethylene (t 1,2‐DCE) was administered (i.p.) in a dose‐response fashion (from 50 to 6.25% of the respective LD50) for three consecutive days, to Swiss Albino CD1 mice and the dealkylation of pentoxyresorufin examined in hepatic microsomes as 2B1 probe. For each hydrocarbon, linear regression was performed for both the inductive and toxic slope related to 2Bl‐like activity in order to evaluate the IC100 [the dose, in mmol/kg, pro...

Correlation between cytochrome p450 2b1 induction by barbiturates and toxicity in the chick embryo in ovo

Using the highly sensitive probe‐substrate pentoxyresorufin towards cytochrome P450 2B1 isozymes, the relationship between the induction of this set of hemoproteins by barbiturates and toxicity was studied. Each chemical, allobarbital (AB), amobarbital (AM), barbital (BA), butobarbital (BB), buthetal (BU), cyclobarbital (CB), heptobarbital (HE), hexobarbital (HX), mephobarbital (ME), pentobarbital (PA), pentotal (PT), phenobarbital (PB), secobarbital (SE) and vinbarbital (VB) was injected in the chick embryo at the 16–18 day of embryonic incubation in equivalent doses (50, 25, 12.5, 6.25 and 3.12% of the respective LD50) and the mixed function monooxygenase activity examined in purified hepatic microsomes. For each barbiturate, linear regression was performed for both the inductive and toxic slope related to P450 2B1‐like activity in order to evaluate the IC100 in μmol/egg [the dose producing a 100 % increase of pentoxyresorufin dealkylation (PROD) during the induction phase] and the TC100, (the dose prod...