Evaluation of immunoassays as an alternative for the rapid determination of pesticides in wine and grape samples.

The main objective of this paper is to address the performance of immunochemical assays for the detection of the residues of three pesticides [atrazine, bromopropylate, and 2,4,6-trichlorophenol (TCP)] in real winery samples, such as wine, grapes, and grape juice. Different approaches have been evaluated to minimize interferences from the matrixes, and suitable working protocols have been established in order to achieve the necessary LODs, accuracy, and precision for real samples. A simple dilution of the sample proved to be sufficient for the determination of atrazine and bromopropylate in red and white wine and grape juice at the required levels of concentration. However, for TCP, an SPE procedure has been optimized using amino cartridges. The recoveries were above 85% in all cases, and the LOD values were below the parts per billion level, except for bromopropylate, which ranged between 2 and 50 microg/L, depending on the matrix. The grape matrix effect could be resolved by a simple extraction with methanol. Complete recoveries were obtained, and the final measurement procedures were able to determine selected pesticides below their maximum residue levels. The newly developed methods have been compared with standard chromatographic methods.

Early hepatic changes induced in rats by two hepatocarcinogenic organohalogen pesticides: bromopropylate and DDT.

The aim of the present studies was to describe the effect of two organohalogen pesticides: DDT and bromopropylate, on early changes in rat liver, proposed in the literature to be useful endpoints in screening of non-genotoxic hepatocarcinogens and/or liver tumor promoters. We investigated the effects on the following endpoints: hepatomegaly, mitogenesis (DNA synthesis, mitotic activity, percentage of binuclear cells) and cytochrome CYP2B1-dependent monooxygenase induction. The histological and cytochemical changes in the liver were also recorded. Male Wistar rats received bromopropylate in one, three or five daily oral doses of 125, 250, and 500 mg/kg body wt. day-1. DDT was applied as one, three, and five daily oral doses of 24 mg/kg body wt. day-1 (this dose is close to the mean hepatocarcinogenic dose in male Wistar rats: 34.1 mg/kg body wt. day-1). In the case of both pesticides the early effects observed consisted of hepatomegaly accompanied by an increase in the p-nitroanisole O-demethylase activity and hepatocyte proliferation. Hepatocyte proliferation was elevated during the total experimental period. Vacuolated cytoplasm and evident focal necrosis may suggest that the maximal increase in hepatocyte proliferation, preceding hepatomegaly, is at least partly related to a regenerative liver response to pesticides. In addition to the above-mentioned early changes, the present findings provide new evidence for the occurrence of dose-dependent abnormal mitoses (and c-mitoses) in the hepatocytes of the bromopropylate and DDT treated rats.

Bromopropylate: induction of hepatic cytochromes P450 and absence of covalent binding to DNA in mouse liver.

Oral administration of benzilic acid ester-based acaricide bromopropylate at daily doses of 3, 15, 100, and 300 mg/kg body wt to young adult male Tif:MAGf mice for 14 days caused slightly increased liver weights in the high-dose group. A dose-dependent increase of the microsomal cytochrome P450 content was accompanied by elevated ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, pentoxyresorufin O-depentylase, and total testosterone hydroxylase activities. When compared with mice treated in parallel with the model compounds for hepatic xenobiotic metabolizing enzyme induction, phenobarbitone, and 3-methylcholanthrene, the enzyme activity changes observed with bromopropylate largely equalled those expressed in phenobarbitone-treated mice. Immunochemical studies with monoclonal antibodies against rat liver cytochrome P450 isoenzymes of the gene families 1A, 2B, 3A, and 4A confirmed that bromopropylate is a phenobarbitone-type inducer in the mouse liver. Titration of liver microsomal suspensions with bromopropylate yielded Type I substrate binding spectra. The specific amplitude was increased 1.5-fold when microsomes from bromopropylate-treated mice (300 mg/kg body wt) were used instead of control microsomes, indicating the induction of cytochromes P450 catalyzing the oxidative metabolism of the test compound. Single oral administration of 300 mg/kg body wt [14C]bromopropylate to male mice, without or following pretreatment for 14 days with 300 mg/kg body wt unlabeled bromopropylate, gave no indication for DNA binding of the test compound in the liver. This excludes a genotoxic potential via covalent DNA modification. The results suggest that, in analogy to phenobarbitone, bromopropylate acts as a tumor promotor rather than a tumor initiator in the mouse liver.

Promotion of altered hepatic foci development in rat liver, cytochrome P450 enzyme induction and inhibition of cell-cell communication by DDT and some structurally related organohalogen pesticides.

The organochlorine pesticide 1,1'-(2,2,2-trichloroethylidene) bis(4-chlorobenzene) (DDT) and four structural analogues (bromopropylate, chlorobenzilate, dicofol and fenarimol) were investigated for their ability to inhibit gap junctional intercellular communication both in the Chinese hamster V79 metabolic co-operation assay and in the scrape-loading/dye-transfer assay in WB-F344 rat liver epithelial cells. The pesticides were also studied for their ability to enhance the development of gamma-glutamyltranspeptidase-positive altered hepatic foci and induce cytochrome P450 monooxygenase isoenzymes in nitrosamine-initiated male Sprague-Dawley rats. The in vitro studies showed all organohalogens except fenarimol to be potent inhibitors of cell-cell communication in both test systems used. Concomitant results were recorded in the in vivo study. Thus, all potent inhibitors of intercellular communication were found to enhance significantly foci development and fenarimol was again without any significant effect. All pesticides studied were shown to be potent inducers of the phenobarbital-inducible cytochrome P450b isoenzyme and to cause hepatomegaly. Thus, no strict correlation between cytochrome P450b induction/liver growth and tumour promotion-related effects in vivo and in vitro was apparent for these organohalogen pesticides in the present study.

Evaluation of pesticides which pose carcinogenicity potential in animal testing. II. Consideration of human exposure conditions for regulatory decision making.

In reaching a regulatory decision on the use of pesticides with carcinogenic potential, it is of great importance to investigate the extent of dermal exposure and absorption of a pesticide to users and field workers. By applying this information, along with the appropriate carcinogenicity categorization of a pesticide, a reasonably sound regulatory decision can be derived. Seven pesticides were selected, based on adequacy of tumor data, and were taken through the tumor evaluation system as reported in Part I 1984, Regul. Toxicol. Pharmacol. 4, 355-360). A step-by-step analysis on how a regulatory decision is reached on each pesticide by the EPA and CDFA was discussed.

Carcinomas of the liver and forestomach in mice ingesting chlorobenzilate.

The maximal tolerated dose of chlorobenzilate was given by continuous oral administration, starting at the age of 7 days, to both sexes of two hybrid strains of mice-(C57BL/6 X C3HAnf)F1 and (C57BL/6 X AKR)F1. There was an increased incidence of tumors in one or more organs in male mice of both strains and female mice of the (C57BL/6 X C3HAnf)F1 strain. Male mice of both strains developed significantly elevated incidences of carcinomas of the liver. Female (C57BL/6 X C3HAnf)F1 strain mice had a significantly increased incidence of hyperplasia and carcinomas of the forestomach.