Comparison of benzo(a)pyrene metabolism by testicular homogenate and the isolated perfused testis of rat following 2,3,7,8-tetrachlorodibenzo-P-dioxin treatment

Benzo(a)pyrene (BP) metabolism was studied in the cell free testicular homogenate and in the isolated perfused rat testis 72 h following tetrachlorodibenzo-P-dioxin (TCDD). The BP concentration for both metabolic systems was 2 × 10^–7 M. BP metabolites were extracted from testicular homogenate, perfusate and testicular tissue and subjected to high-pressure liquid Chromatographic analysis. The ratio of various BP metabolites in the cell free homogenates ranged from 3.5 to 164 times those of the isolated perfused testis, and the total BP metabolites in the cell free system of either control or TCDD-induced testis were 16 times that of the intact isolated perfused testis. The major BP metabolites in the organic extractable phase from the isolated perfused testis and the testicular homogenate were BP dihydrodiols and BP phenols, respectively.The ratio of water soluble metabolites to organic soluble metabolites in the homogenate and the isolated perfused testis is 1.1 and 3.0 respectively. Therefore, in the intact isolated testis, water soluble BP metabolites are formed three times greater than those of the organic soluble BP metabolites, and thus suggests that specific conjugating enzyme activities in the intact testis are greater than those of the homogenates. The magnitude of various BP metabolites in either the homogenates or the isolated perfused testis of TCDD treated rats ranged from 1.4 to 2.2 times their respective controls, except 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene in the isolated perfused testis was not altered by TCDD treatment. In conclusion, the isolated perfused rat testis is metabolically active and capable of biotransforming PAH. This system better reflects metabolic capability of the intact organ in the rats than do cell free homogenate system. The isolated perfused testis system retains the integrity of the complex biological organization of tissues, cell types, and enzymes in testicular metabolism and may provide data that may aid in the prediction of germ cell mutation as well as toxicity.The Abbreviations Used are PAH polycyclic aromatic hydrocarbons - BP benzo(a)pyrene - 9,10-diol 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene - 7,8-diol 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene - 4,5-diol 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene - 3-OH 3-hydroxybenzo(a)pyrene - 9-OH 9-hy-droxybenzo(a)pyrene - 7-OH 7-hydroxybenzo(a)pyrene - 12-OH 12-hydroxybenzo(a)pyrene - 1,6-quinone benzo(a)pyrene-1,6-dione - 3,6-quinone benzo(a)pyrene 3,6-dione - 6,12-quinone benzo(a)pyrene 6,12-dione - DMBA 7,12-dimethylbenzanthracene - DMN dimethylnitrosamine - 7,8-diol 9,10-epoxide 5,7-t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene - HPLC high pressure liquid chromatography - TCDD 2,3,7,8-tetrachlorodibenzo-P-dioxin - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - NADP⁺ nicotinamide adenine dinucleotide - AHH aryl hydrocarbon hydroxylase - EH epoxide hydrolase - GSH-T glutathione transferase - HPRT hypoxanthine phosphoribosyltransferase