Laura Fabrizi

Kinetic parameters of OPT pesticide desulfuration by c-DNA expressed human CYPs

The role of different cytochrome P450 isoforms (CYPs) in the desulfuration of four organophosphorothionate pesticides (OPTs), namely diazinon (DIA), azinphos-methyl (AZ), chlorpyrifos (CPF) and parathion (PARA), at OPT levels representative of actual human exposure has been investigated. For this purpose c-DNA expressed human CYPs and a method, based on acetylcholinesterase (AChE) inhibition, able to detect nM levels of oxon have been used. Our results indicate that the four tested OPTs at low concentration were mainly desulfurated by CYP2B6, 2C19 and 1A2, showing K(m) values in the range 0.8-5 μM and the highest efficiency (intrinsic clearance (ICL)) values. CYP3A4 was generally endowed with high K(m) and resulted linear up to 25-100 μM OPT, concentrations saturating the most efficient CYPs. The tentative extrapolation of the relative contribution of single CYPs, taking into account the average content of different isoforms in the human liver, indicate that CYP1A2 is the major responsible for oxon formation. Indeed this CYP catalyses the 50-90% of desulfuration reaction, depending on the OPT. As CYP3A4 activity is not completely saturated up to 100 μM OPT, and due to the high hepatic content, its contribution to oxon formation may result relevant in poisoning episodes, when individuals are exposed at high doses of OPTs.

Identification of the cytochrome P450 isoenzymes involved in the metabolism of diazinon in the rat liver.

The metabolism of diazinon, an organophosphorothionate pesticide, to diazoxon and pyrimidinol has been studied in incubations with hepatic microsomes from control Sprague–Dawley (SD) rats or SD rats treated with different P450‐specific inducers (phenobarbital, dexamethasone, β‐napthoflavone, and pyrazole).

Mechanistic aspects of organophosphorothionate toxicity in fish and humans.

Metabolic transformation plays a major role in the mechanism of toxicity of organophosphorous (OP) pesticides. The modulation of their toxicity by oxonases and monooxygenases, alone or in combination, has been shown in mammals and fish. Very limited information exists for the identification of the metabolic factors relevant in the human toxicology of such chemicals. In this paper, we develop a simple algorithm, based on in vitro data, for the identification of fish species more susceptible to diazinon (D). Similar algorithms are likely to be applicable to other organophosphothionate (OPT) pesticides. We also report on preliminary studies on the OPT substrate specificity of human liver cytochromes P450 (CYPs): such information may be useful to understand the role of sulphoxidation in OPT toxicity to humans and to identify individuals with increased susceptibility to OPT toxicity. Studies of the mechanism of OPT toxicity may provide useful tools for a more detailed characterisation of these chemicals, with reference to the risk for the human population and to the impact on the fish species present in specific environments.

Long Depletion Time of Enrofloxacin in Rainbow Trout (Oncorhynchus mykiss)

ABSTRACT The international production of farmed fish has been growing continuously over recent years. Until now few veterinary drugs have been approved by the European Union for use in aquaculture, and this has favored the off-label use of products authorized for use in food-producing animal species different from fishes among fish farmers. Adequate field studies are lacking, especially for those species called minor species which are consumed extensively only in some European countries. In the present investigation we studied the depletion of the fluoroquinolone antibacterial enrofloxacin over time in a minor species, the rainbow trout (Oncorhynchus mykiss), reared on a real fish farm and treated with medicated feed (10 mg kg of trout body weight−1 day−1). Edible tissue samples (muscle plus skin in natural proportions) and fish bone samples were analyzed for enrofloxacin and for its major metabolite, ciprofloxacin, by high-performance liquid chromatography with fluorescence detection at different times after the end of treatment. Our results show that at 500°C-day (in which degree-days are calculated by multiplying the mean daily water temperature by the total number of days on which the temperature was measured), which is the minimum withdrawal period established by European Economic Commission Directive No. 82/2001 for any type of product administered off-label, edible trout tissues might still contain about 170 μg of enrofloxacin kg−1, whereas the maximum residue level for enrofloxacin plus ciprofloxacin is set at 100 μg kg−1. To our knowledge, no studies of the depletion of enrofloxacin in rainbow trout have been performed. On the basis of the data obtained in the present study, we suggest a more appropriate withdrawal time of 816°C-day for the sum of enrofloxacin plus ciprofloxacin levels in rainbow trout muscle plus skin tissues.

The role of different cytochrome P450 isoforms in in vitro chloroform metabolism

The two CHCl3 activation pathways have been studied in incubations at different oxygenation conditions with hepatic microsomes from control Sprague Dawley (SD) rats or SD rats treated with different cytochrome P450 inducers (acetone, phenobarbital, pyrazole, dexamethasone, and beta-naphthoflavone). The present results provide direct evidence that CHCl3 concentration is critical in determining the role of different cytochrome P450 isoforms (CYP) and the related effects of metabolic inducers. At 0.1 mM CHCl3 concentration, the only major contribution to its oxidative biotransformation in liver microsomes from untreated rats was due to CYP2E1, as shown by metabolic inhibition due to 4-methylpyrazole or by anti-CYP2E1 antibodies. Moreover, animal treatments with acetone and pyrazole increased the production of adducts of phosgene to microsomal phospholipid by about 10-15 times. At 5 mM chloroform, in control rat liver microsomes, CYP2B1/2 was the major participant responsible for chloroform activation, while CYP2E1 and CYP2C11 were also significantly involved. Consistently, at this chloroform concentration, the effect of phenobarbital (CYP2B1/2 inducer) was maximal, producing very high levels of adducts. The reductive pathway was expressed at 5 mM CHCl3 only and was not significantly increased by any of the inducers used. Moreover, it was not inhibited by metyrapone and 4-methylpyrazole or by anti CYP2C11 antibodies. Therefore, it may be concluded that, in the range of chloroform concentrations tested, those CYPs involved in CHCl3 oxidative bioactivation do not participate in CHCl3 reduction. Chloroform oxidative metabolism in PB-microsomes could achieve very high absolute rates, much higher than those in C-microsomes; in contrast, the metabolic rates in AC- and PYR-microsomes remained within the activity levels observable in C-microsomes at high chloroform concentration. Therefore, it can be argued that the CYP2B1/2-mediated induction of CHCl3 activation is the basis for the effect of PB in potentiating chloroform hepatotoxicity. Moreover, processes other than CYP2E1-mediated metabolic induction may be more relevant in the ketones potentiation of chloroform-induced acute toxicity.

Orally Administered Erythromycin in Rainbow Trout (Oncorhynchus mykiss): Residues in Edible Tissues and Withdrawal Time

ABSTRACT Aquaculture production has notably increased in the last decades, mainly thanks to intensive farming. Together with market globalization, this gives rise to the spreading of several fish diseases, thus increasing the demand for veterinary drugs for aquatic species. Nonetheless, very few chemicals are registered for use in aquaculture, and fish farmers are often forced to resort to off-label use of drugs authorized for other food-producing animal species. Rainbow trout is the major farmed fish species in Italy and the second one in Europe. Erythromycin is the antibiotic of choice against gram-positive cocci, the major concern for trout farming, but it is not yet registered for aquaculture use in most European countries. The aim of this study was to follow the depletion of erythromycin in rainbow trout (Oncorhynchus mykiss), after its administration at 100 mg kg−1 trout body weight day−1 for 21 days through medicated feed (water temperature, 11.5°C). Erythromycin residues in fish muscle plus skin in natural proportion were determined by a validated liquid chromatography-electrospray ionization-tandem mass spectrometry method. Interpolation of our data, following European Agency for the Evaluation of Medicinal Products guidelines, gives a withdrawal time of 255°C-days (°C-day = water temperature × days), thus showing that the general value (500°C-day) recommended by the Council Directive (EEC) no. 82/2001 for off-label drug use in aquaculture would be too conservative in this case, with excessive costs for the farmers. Our study provides preliminary data for a more prudent use of erythromycin in rainbow trout, suggesting a possible withdrawal time after treatment.

Time dependence of chloroform-induced metabolic alterations in the liver and kidney of B6C3F1 mice

Abstract The time course of some biochemical changes in the liver and in the kidney was studied in B6C3F1 male mice dosed with a single i.p. injection of 150 mg/kg body weight (b.w.) CHCl3. Hepatic and renal microsomal cytochrome P450 (P450) content and some related monooxygenase activities, CHCl3 oxidative and reductive metabolism, cytosolic reduced glutathione (GSH) content and serum markers of nephrotoxicity were measured. In the liver no biochemical changes were produced up to a week after chloroform treatment. On the contrary, the drug-metabolizing enzyme system in the kidney was dramatically and rapidly inactivated by chloroform treatment. Maximum loss of GSH (50%), P450 (80%) and of different enzymatic activities, including CHCl3 bioactivation, occurred during the first 5 h. These biochemical alterations are early effects, not secondary to morphological tissue changes. Kidney parameters, altered by chloroform treatment, returned to control values at different times: renal function markers became normal in 48 h; GSH levels were recovered at 96 h and the drug-metabolizing enzyme activities at longer times. The present results clearly show that repeated daily doses of chloroform, as those used in carcinogenicity tests, find renal tubular cells not at their physiological status, due to the changes produced by the first chloroform dose. Therefore the similarity in P450-dependent chloroform metabolism shown in vitro by hepatic and renal microsomes from untreated B6C3F1 male mice or in vivo in animals treated once, is lost during repeated treatments. These features should be considered in understanding the different susceptibility of the liver and the kidney to chloroform-induced tumours.

Rapid depletion of marbofloxacin residues in rabbit after therapeutic treatment.

Although rabbit meat production represents a very small percentage of the world meat market, this percentage has been growing continuously during the last 30 years. Rabbit is considered a minor food species, and therefore no drugs are specifically registered for this animal. This situation encourages rabbit farmers to make off-label use of antibacterial drugs authorized for food-producing animal species other than rabbits. In the present study, the distribution and elimination of the fluoroquinolone antibacterial agent marbofloxacin in rabbit muscle, liver, and kidney was investigated. Marbofloxacin was chosen as a representative of a new generation of antibacterial drugs active against most gram-positive and gram-negative bacteria and mycoplasms; it is well tolerated and has short elimination times in bovine and swine species. Rabbits were treated with marbofloxacin at 2 mg kg of body weight(-1) for 5 days. Residual concentrations in liver, kidney, and muscle tissues were determined posttreatment with high-performance liquid chromatography and fluorescence detection. Marbofloxacin was rapidly distributed and eliminated from rabbit tissues. Concentrations were higher in the liver and kidney than in muscle. However, 48 h after the end of treatment, marbofloxacin concentrations dropped below the maximum residue level fixed for this antibacterial drug in cattle and pigs. Considering the efficacy of marbofloxacin for the treatment of the most common rabbit diseases, its tolerability, and its short elimination time as verified in the present study, use of this antibacterial drug could be extended to therapeutic treatment of rabbits.