Influence of DNA repair polymorphisms on biomarkers of genotoxic damage in peripheral lymphocytes of healthy subjects.

DNA repair polymorphisms may represent susceptibility factors affecting DNA integrity, and possibly cancer risk, in human population. In order to elucidate the influence of a few widely studied DNA repair polymorphisms on individual levels of DNA damage and their possible interaction with lifestyle and environmental exposures, 171 subjects from a well-characterized human population enrolled in a previous study on genetic effects of air pollution were genotyped for the XRCC1 Arg280His and Arg399Glu, XRCC3 Thr241Met and ERCC2 Lys751Gln polymorphisms. The association between DNA repair genotype, alone or in combination with metabolic genotype, on the levels of SCE, micronuclei and tail moment values in peripheral lymphocytes was evaluated. A significant influence of the ERCC2 genotype on SCE frequency was observed. Subjects with ERCC2 751 Gln/Gln genotype had significantly higher risk of high (above the median) SCE/cell with respect to Lys/Lys referents (OR 4.55, 95% CI 1.48-13.99). A non-significantly elevated OR was also observed in Gln/Lys heterozygotes, suggesting a gene dosage effect. When subjects were categorized by smoking habits and professional exposure, the variant ERCC2 751 Gln/Gln genotype was associated with elevated SCE rates in non-smokers and in exposed subjects, but not in smokers. The results of this study support the hypothesis that some DNA repair polymorphisms exert a modifying effect on individual levels of DNA damage in healthy subjects, possibly also modulating cancer risk.

Cox and mesothelioma: an overview.

Cyclooxygenases catalyze the rate limiting step in the production of prostanoids. Accumulating data demonstrate that overexpression of these enzymes, and in particular of cyclooxygenases-2, promotes multiple events involved in tumorigenesis; in addition, numerous studies show that inhibition of cyclooxygenases-2 can delay or prevent certain forms of cancer. Malignant mesothelioma is a lethal pleural, peritoneal and pericardial neoplasia that actually lacks valid therapies and in which cyclooxygenases-2 is recognized as an important adverse prognostic factor. Hence, there is an increasing interest in the development of new treatments based on cyclooxygenases-2 inhibitors, to prolong survival and even potentially cure this neoplasia.

Production of antibodies against the coenzyme pyrrolequinoline quinone.

Polyclonal antibodies against pyrrolequinoline quinone have been elicited in rabbits. These antibodies react with free and protein-bound pyrrolequinoline quinone. In particular they react with native and denatured lentil seedling amine oxidase as detected by dot-blot and ELISA assays. The presence of 1 mol pyrrolequinoline quinone per mol of enzyme was determined by the last method.

Modulation of DNA binding in vivo by specific humoral immunological response: a novel host factor in environmental carcinogenesis?

To investigate the possible modulatory effect of the immune response induced by recurrent carcinogen exposure, a specific humoral immune response toward 2-acetylaminofluorene (2-AAF) was elicited in Swiss mice with repeated intraperitoneal injections of a 2-AAF-gelatin conjugate. The immunization procedure resulted in the production of specific anti-2-AAF antibodies in all treated animals. Groups of immunized and nonimmunized mice were subsequently fed 2-AAF pelleted in the diet at 50 and 150 ppm for 4 weeks. At the end of 2-AAF administration, animals were sacrificed and the content of 2-AAF-adducts in liver DNA was determined by enzyme-linked immunoadsorbent assay using a polyclonal rabbit antiserum. The comparison of the adducts levels in immunized and nonimmunized mice (receiving either the vehicle or the adjuvant alone during pretreatment) demonstrates a highly significant (p < 0.001) difference among groups, with far lower adduct levels in immunized animals. No significant difference in food consumption or liver metabolic activities was observed among experimental groups, suggesting the absence of external bias. The mechanism underlying the result observed is not yet clear; however, the experimental data strongly suggest that the specific immunological response induced by recurrent carcinogen exposure may exert a modulatory effect and act as a relevant host factor in chemical carcinogenesis.

Analysis of micronuclei in peripheral blood lymphocytes of traffic wardens: effects of exposure, metabolic genotypes, and inhibition of excision repair in vitro by ARA-C.

The cytokinesis-block micronucleus (MN) assay in peripheral lymphocytes was used to assess the genetic effects of the occupational exposure to traffic fumes in policemen from the Municipality of Rome. The study population consisted of 192 subjects engaged in traffic control (exposed, 134 subjects), or in office work (controls, 58 subjects). Groups were balanced for age, gender, and smoking habits. The average benzene exposure during the workshift was 9.5 and 3.8 microg/m(3) in exposed individuals and controls, respectively. All subjects were genotyped for CYP1A1, CYP2E1, GSTM1, GSTT1, and DT-diaphorase polymorphisms. The incidence of micronuclei and micronucleated cells was recorded in 1,000 binucleated cells harvested 66 hr after mitogen stimulation. Regression analysis of data showed that MN frequency was mainly modulated by the age (P = 0.001) and gender (P = 0.001) of the study subjects (relatively higher in the elderly and females), whereas it was unaffected by the occupational exposure to traffic fumes and smoking habits. A weak (P = 0.02) association between lower MN frequency and the GSTM1 null genotype was also observed. In order to improve the sensitivity of the method to excision-repairable lesions, a modified protocol, with exposure of cells to the repair inhibitor cytosine arabinoside (Ara-C) during the first 16 hr of growth, was applied to 78 subjects (46 exposed and 32 controls). The results confirmed the higher MN frequency in females (P < 0.05), but failed to demonstrate any significant effect of chemical exposure (occupational or related to smoking habits). When the frequency of MN induced by Ara-C (i.e., spontaneous values subtracted) was considered, a significant inverse correlation with age was observed (P = 0.005), possibly related to the age-dependent decrease in repair proficiency.

Effects of specific humoral immunity on DNA-adduct formation in Swiss mice treated with benzo[alpha]pyrene.

In order to study the effect of possible modulating factors on DNA-binding carcinogens, we investigated the role of specific immune response on racemic 7,t-8-dihydroxy-t-9,10-epoxy-7,8,9, 10-tetrahydro-benzo[a]pyrene ((+/-)-anti BPDE)-DNA (BPDE-DNA) adduct formation. Anti BPDE Immunoglobulin G (IgG) were elicited in Swiss mice before subsequent carcinogen administration. The immunization schedule consisted of four weekly subcutaneous injections of both (+/-)-anti BPDE-gelatin (BPDE-Gel) and DNA (BPDE-DNA) conjugate, followed by a final immunogen injection 14 days later. The immunization procedure resulted in the production of specific anti-BPDE antibodies in all treated animals. One week after the end of the immunization procedure, both groups of immunized and non immunized mice were treated with different doses of B[a]P (25-50-100-200 mg B[a]P/Kg body weight) by intraperitoneal injection. Seven days after treatment, the mice were sacrificed. Adduct levels were detected by competitive ELISA by using optimal conditions established in our laboratory and highly specific and sensitive IgG anti BPDE-DNA induced in rabbit. The determination of DNA adducts in liver revealed significantly lower B[a]P adduct levels in liver of immunized mice with respect to non-immunized animals. This result confirms those obtained for 2-acetylaminofluorene (2-AAF) in a previous work: the specific humoral immunity elicited by repeated carcinogen exposure may be able to modulate the genotoxic effect induced by subsequent carcinogen administration.

Quantitation of adriamycin content by a sensitive immunochemical assay.

An immunoenzymatic method for the quantitation of adriamycin (ADM) content in tissues as well as in tumor cells has been developed. This procedure has three main advantages. Firstly, it is possible to carry out the determination on whole homogenates and blood serum, thus avoiding the extractive procedures. Secondly, very low ADM concentrations (0.2 ng) can be detected. Thirdly, it is possible to determine simultaneously and in triplicate both the standard curve and ADM concentration in twelve different samples with a great reduction of the experimental variability.

Identification of optimal conditions for the detection of benzo[a]pyrene-DNA adducts by enzyme-linked immunoadsorbent assays (ELISA).

The aim of the present report was to establish the optimal conditions for the detection of polycyclic aromatic hydrocarbon adducted to DNA by enzyme-linked immunoadsorbent assays (ELISA). Racemic 7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene ((+/-)-anti-BPDE) modified DNA samples were produced in vitro, by reacting (+/-)-anti-BPDE with calf thymus DNA, and in vivo in Swiss female mice by single i.p. injection of benzo[a]pyrene (B[a]P) (200 mg/kg body weight dissolved in tricaprylin). The BPDE adduct content in vitro and in liver and lung modified DNA was detected by direct and competitive ELISA using serial dilutions of the samples in unmodified calf thymus DNA, and polyclonal rabbit immunoglobulin-G elicited toward BPDE-DNA and BPDE-gelatin, both produced in our laboratory. The carcinogen-macromolecule conjugate in which adducts were sought could be used as an immunogen to produce a specific and potent antibody. Moreover, the modification level of the ELISA standards should be as close to the range as of the biological samples to correctly calculate the adducts, since different binding efficiency between antibody and BPDE-modified DNA is dependent on the BPDE modification level (33). Appropriate extraction of the in vitro modified samples is also necessary to guarantee the exact covalent modification level, eliminating noncovalently linked BPDE. Under these conditions, our results confirm that competitive ELISA is much more sensitive than the direct method, mainly because of the limitations caused by the coating of the antigen in each well (max 5 micrograms DNA/well), whereas the amount of DNA (modified or not) that can be employed for adduct detection by competitive ELISA increases 20-fold. The sensitivity obtained was 0.5 fmol B[a]P/microgramDNA (1.6 adducts/10(7) nucleotides).

Immunochemical determination of lonidamine in rat tissues and blood serum.

Lonidamine (LND) is an antitumor drug which interferes selectively with the energy metabolism of neoplastic cell. Because of its physico-chemical properties, LND determination with conventional methods, i.e. high performance liquid chromatography and spectrofluorimetry, gives rise to several problems: LND is a lipotropic drug which becomes intimately associated with biological membranes so that it is impossible to extract all the drug bound, thus leading to an underestimation of the LND content in cells and tissues. These difficulties can be overcome by the immunoenzymatic method described here. The assay is simple, rapid, practical, highly sensitive (2-5 ng/ml) and particularly suitable for the analysis of multiple samples (twelve samples in triplicate for each plate). There is, moreover, a great improvement in data reproducibility.

Biomonitoring of exposure to urban air pollutants: analysis of sister chromatid exchanges and DNA lesions in peripheral lymphocytes of traffic policemen.

In order to elucidate the health effects of occupational exposure to traffic fumes, a few biomarkers of early genetic effect were investigated in Rome traffic policemen. One hundred and ninety healthy subjects engaged in traffic control (133 subjects) or in office work (57 subjects) participated the study. For all subjects, detailed information on smoking habits and other potential confounders were recorded by questionnaires. Average exposure of the study groups to benzene and other aromatic hydrocarbons was evaluated in a parallel exposure survey. All workers were genotyped for the following metabolic polymorphisms: CYP1A1 (m1, m2, and m4 variants), CYP2E1 (PstI and RsaI), NQO1 (Hinf1), GSTM1 and GSTT1 (null variants). In this paper, the results of the analysis of sister chromatid exchanges (SCE) in peripheral lymphocytes, and DNA damage by alkaline (pH 13) comet assay in mononuclear blood cells are reported. No statistically significant difference in the frequency of SCE or high frequency cells (HFC) was observed between traffic wardens and office workers (controls), despite the significantly higher exposure to benzene of the former (average group exposure 9.5 versus 3.8microg/m(3), 7h TWA). Conversely, both SCE per cell and HFC were highly significantly (P<0.001) increased in smokers compared to nonsmokers, showing a significant correlation (P<0.001) with the number of cigarettes per day. Multiple regression analyses of data, with metabolic polymorphisms, smoking habits, alcohol consumption, age, gender, and family history of cancer as independent variables, showed that smoking habits, and possibly the CYP2E1 variant genotypes, were the main factors explaining the variance of both SCE and HFC. Within smokers, an association of borderline significance between the CYP1A1 variant genotypes and increased SCE (P=0.050) and HFC (P=0.090) was found. This effect was mainly observed in light smokers (<15 cigarettes per day). The analysis of DNA damage by comet assay did not highlight any statistically significant difference between the exposed and control workers. Moreover, no significant model explaining tail moment variance was obtained by multiple regression analysis using the independent variables shown above. On the whole, these results indicate that exposure to moderate air pollution levels does not result in a detectable increase of genetic damage in blood cells. This evidence does not rule out any possibility of adverse effects, but strongly suggests that in urban residents life-style related factors, such as tobacco smoking, give the prevailing contribution to individual genotoxic burden.

Effects of prenatal AZT on mouse neurobehavioral development and passive avoidance learning.

Recent evidence has shown that perinatal administration of zidovudine (AZT) to HIV-infected mothers reduces the risk of maternal-infant transmission of the virus. Treatment of pregnant seropositive women with AZT is becoming a common medical practice, despite the paucity of information about the potential neurotoxic/behavioral-teratogenic effects of AZT on the developing organism. The aim of the present study is to evaluate in mice the short-, medium-, and long-term effects of prenatal exposure to AZT on neurobehavioral development. Pregnant mice were given 0.2, 0.4, and 2.0 mg/ml AZT in drinking water from day 10 of gestation to delivery. Offspring's viability was severely affected in the 2.0 mg/ml AZT group. Thus, behavioral analysis was carried out in offspring of 0.2 and 0.4 mg/ml AZT-treated females only. Some limited but significant alterations were found, such as stunted body weight, delayed appearance of the pole-grasping reflex, and a slight impairment in the acquisition phase of a passive avoidance response. Moreover, sexual differences in some items of the social behavior repertoire appeared to be affected by AZT treatment.

Metabolic polymorphisms and urinary biomarkers in subjects with low benzene exposure.

The effect of some common metabolic polymorphisms on the rate of trans,trans-muconic acid (TMA) and S-phenylmercapturic acid (SPMA) excretion was investigated in 169 policemen exposed to low benzene levels (<10 microg/m3) during the work shift. End-shift urinary concentrations of TMA and SPMA, normalized to unmetabolized blood benzene concentration, were used as indicators of individual metabolic capacity. CYP2E1, NQO1, GSTM1, and CSTT1 polymorphisms were analyzed in all subjects by polymerase chain reaction (PCR) restriction fragment length (RFL). The results obtained show significantly elevated levels of TMA and SPMA in urine of smokers compared to nonsmokers, whereas no correlation with environmental benzene was observed. TMA/blood benzene ratio was partially modulated by glutathione S-transferase (GST) genotypes, with significantly higher values in null individuals (GSTM1 and GSTT1 combined). However, a greater fraction of total variance of TMA/blood benzene in the study population was explained by other independent variables, that is, season of sampling, smoking habits, and gender. Variance in SPMA/blood benzene ratio was only associated with smoking and occupation, whereas no significant role was observed for the metabolic polymorphisms considered. These results suggest that in a population exposed to very low benzene concentrations, urinary TMA and SPMA levels are affected to a limited extent by metabolic polymorphisms, whereas other factors, such as gender, lifestyle, or other confounders, may account for a larger fraction of the interindividual variability of these biomarkers.

Search of anti-adriamycin antibodies in serum of cancer patients under chemotherapic treatment.

The exposure to DNA reactive carcinogens is known to elicit a specific humoral immunological response, with the production of antibodies towards the carcinogen adducts. In analogy to chemical carcinogens, any chemotherapic, like Adriamycin, undergoes the same adduct formation, and for this reason could elicit specific antibodies. In this case we can suppose that an eventual immunological response could influence the efficacy of chemotherapy. The aim of this study was to verify if adriamycin adducted to DNA or transport proteins can elicit an immunological response of specific anti-adriamycin (ADM) antibodies in sera of 43 cancer patients treated with the drug. No specific antibodies were detected in these individuals. The lack of anti-adriamycin antibodies suggests that the therapeutic exposure to the drug does not elicit a specific immunological response.

Detection of antibodies to the benzo(a)pyrene diol epoxide-DNA adducts in sera from individuals exposed to low doses of polycyclic aromatic hydrocarbons.

The exposure to DNA reactive carcinogens is known to elicit a specific humoral immunological response, with the production of antibodies toward the carcinogen adducts. Consequently, the presence of circulating anti-carcinogen antibodies has been proposed as a marker of carcinogen exposure, and as a potential modulating factor in chemical carcinogenesis. In this work, the presence of serum antibodies to 7beta,8alpha-dihydroxy-9alpha10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-DNA (BPDE-DNA) adducts was determined in two groups of workers occupationally exposed to low doses of polycyclic aromatic hydrocarbons (PAHs), i.e. policemen (194 subjects) and workers in the aluminum industry (105 subjects). Specific anti BPDE-DNA antibodies were detected in 5.7% (11/194) of policemen and 13.3% (14/105) of aluminium industry workers. Among policemen, a small, not significant (p=0.09), prevalence of positives was observed in traffic wardens compared to office workers. A borderline significant (p=0.052) prevalence of positives was also observed in heavy smokers compared to light smokers among aluminium industry workers. These results basically support previous findings on the association between chronic exposure to polycyclic aromatic hydrocarbons and formation of anti-BPDE-DNA antibodies, even though such association appears to be weak, possibly biased by individual factors which are still largely unidentified.

Divergent synergic effects in carcinogenesis initiation by simultaneous exposure to two genotoxic carcinogens.

Carcinogenesis is a complex and multistep process starting from initiation to tumor progression. Synergistic mechanisms can occur at every step of the process. The aim of this work was to provide information about the effect of chemical carcinogens which, if administered in combination, result in positive as well as negative synergistic effects. In order to evaluate whether for some carcinogens synergism occurs at the initiation step, we compared the effects of Ethylmethanesulfonate (EMS) on Benzo[a]pyrene (BP)-DNA adducts formation in the liver and lung of male Swiss mice treated for seven days by i.p. dose of EMS (1.2 mg/Kg b.w.) alone or by simultaneous administration of three doses of BP (25, 50, 100 mg/Kg b.w.) injected i.p. or the first day of treatment. A group of Swiss mice was treated by BP alone. At it was demonstrated in our laboratory that previous immunization toward BP influences the adduct levels of this carcinogen (14), the same treatments (BaP alone and BaP with EMS) were carried out in mice previously immunized toward BP. Liver and lung 1 BP-DNA adducts were detected in all the groups treated by both BP and EMS as compared to the group treated with BP alone. The EMS-BP association in non-immunized mice showed an antagonistic effect in the liver and a synergistic effect in the lung. In immunized mice a synergistic effect was obtained in both liver and lung. Moreover, the efficiency of both the synergistic and antagonistic effect, depended on BP dose of treatment. It is reasonable to draw the conclusion that simultaneous exposure to BP and EMS leads to different organ-specific and dose-dependent effects. This first preliminary result showed that the pattern of the interaction between genotoxic carcinogens is more complex that was foreseen, even at the stage of DNA adducts formation.

The effect of humoral immunity against adducted benzo[a]pyrene on DNA damage elicited by acute carcinogen exposure in Swiss mice.

Immunoglobulins G (lgG) specific for benzo[a]pyrene-DNA adducts were elicited in Swiss mice by repeated subcutaneous injections of a high molecular weight benzo[a]pyrene-DNA conjugate-adjuvant mix. The immunization procedure resulted in the production of specific antibodies against adducted benzo[a]pyrene B[a]P in all treated animals. One week after completion of the immunization procedure, groups of ten immunized and ten non immunized female mice were treated by single intraperitoneal injection with two different doses of B[a]P. The mice were sacrificed 48 hours after treatment, and both liver and bone marrow cells were isolated for subsequent determinations of DNA binding and micronucleus induction, respectively. Covalent benzo[a]pyrene adducts in liver DNA were detected by competitive ELISA and the incidence of micronucleated polychromatic erythrocytes was evaluated by scoring one thousand cells per animal. The determination of DNA adducts in liver revealed significantly (p < 0.05) lower levels of B[a]P adducts in immunized mice compared to non-immunized animals at both doses, whereas no significant difference was observed between controls. Administration of benzo[a]pyrene produced moderate, dose-related increases in the incidence of micronucleated polychromatic erythrocytes in all treated groups, with no significant difference between immunized and non-immunized mice. The decrease of covalent DNA adducts in the liver of immunized mice suggests that the specific humoral immunity elicited by repeated carcinogen exposure may act as a relevant modulating factor in chemical carcinogenesis.

Activation of 2,4-diaminotoluene to proximate carcinogens in vitro, and assay of DNA adducts.

1. 2,4-Diaminotoluene, which yields adducts with DNA in vivo, has been studied for its ability to form adducts in vitro. Metabolic activation with rat liver post-mitochondrial supernatant gave 300 adducts/10(6) nucleotides in calf thymus single-stranded DNA, under defined experimental conditions. 2. 2,4-Diaminotoluene-modified DNA and deoxyhomopolymers showed characteristic u.v. absorption spectra, exhibiting hyperchromic effects at 235 and 220 nm, and hypochromic effect at 260 nm. The difference spectra between diamine-modified and untreated DNA, or deoxyhomopolymer, were very similar to the spectrum of 2,4-diaminotoluene alone. 3. 2,4-Diaminotoluene-modified DNA was assayed by ELISA with specific monoclonal antibodies directed against diamine-DNA adducts. Reactions with poly-d(A) or poly-d(A-T) gave no spectral modification, and immunochemical analysis showed that the diamine did not bind to these polynucleotides. On the other hand, in the case of poly-d(G) or poly-d(C-G), strong immunoreactions were observed, demonstrating that the guanine base is involved in the binding of the diamine to DNA. 4. Monoclonal antibodies directed against different diamine-DNA adducts have shown that 80% of the in vitro metabolic activation involves the para amino group of the aromatic diamine.

Induction of humoral immunity toward 2-acetylaminofluorene in mice: modulation of DNA binding after 4 weeks dietary exposure to the carcinogen.

In order to investigate the modulatory effect of the immune response induced by recurrent carcinogen exposure, anti-2-acetylaminofluorene (anti-2-AAF) IgG were elicited in Swiss mice before subsequent carcinogen administration. The immunization schedule consisted of three weekly i.p. injections of 2-acetylaminofluorene (2-AAF)-gelatin conjugate, followed by a final immunogen injection 14 days later. At the end of treatment, the presence of specific anti-2-AAF antibodies in blood serum of all immunized animals was demonstrated. The immunization procedure did not affect liver metabolic activities, as evaluated using liver homogenates for the exogenous activation of 2-AAF to mutagen. After immunization, mice were fed 2-AAF pelleted in the diet at 50 and 150 p.p.m. for 4 weeks and killed at the end of treatment. The determination of DNA adducts by ELISA in liver and spleen of treated animals revealed significantly (P < 0.01-0.001) lower 2-AAF adduct levels in both tissues of immunized mice with respect to non-immunized animals (both naive and pretreated with the adjuvant alone). This result suggests that the specific humoral immunity elicited by repeated carcinogen exposure may be able to modulate the genotoxic effect induced by subsequent carcinogen administration.

Characterization of a new high-temperature-induced 66-kDa heat-shock protein, antigenically related to heat-shock protein 72.

M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major "classic" heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45 degrees C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)+ RNA extracted from cells treated at 45 degrees C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)+ RNA extracted from cells heated at 42 degrees C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro. hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer.

Xenobiotic-metabolizing enzyme systems in test fish--II. The ethylmorphine N-demethylase activity of guppy (Poecilia reticulata) liver.

The oxidative demethylation of the model substrate ethylmorphine has been characterized for the first time in the liver of a fish (Poecilia reticulata). The enzyme showed maximal activity at 35 degrees C and pH values higher than 8. The values of Km and Vmax for the reaction were 0.83 +/- 0.11 mM and 4.64 +/- 0.81 nmol HCHO/(mg microsomal protein) per min. The activity is attributed to the cytochrome P-450-dependent monoxygenase system, since it is inhibited by CO and requires NADPH; moreover it is inhibited competitively by alpha-naphthoflavone and non-competitively by metyrapone. The enzyme activity is induced by a two-week treatment of fish with phenobarbital and may be associated with a protein band of Mr 54,000.