Robert D. Combes

Interaction with microsomal lipid as a major factor responsible for S9-mediated inhibition of 1,8-dinitropyrene mutagenicity

1,8-Dinitropyrene (1,8-DNP), present in polluted air, is a rodent carcinogen and a potent, direct-acting mutagen in salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of mammalian hepatic S9 or microsomes. We demonstrate that at least a substantial part of this effect is attributable to non-enzymatic processes. The microsomal-dependent inhibition was unaffected by omission of an NADPH-generating system or when the cytochrome P-450 inhibitor, SKF-525A, or the cytochrome P-448 inhibitor, ellipticine, was incorporated in the metabolic activation system, suggesting that mixed function oxidases are not involved. Heat inactivation partially decreased the ability of induced S9 to reduce DNP mutagenicity. Substitution of S9 with a similar concentration of bovine serum albumin did not affect DNP activity. Thus non-specific binding to microsomal protein is not involved. However, when lipids, derived from uninduced microsomes, were added to incubations of DNP and S. typhimurium TA98, mutagenicity was decreased. Furthermore, substitution of microsomal lipids with a suspension of phosphatidylcholine (PC), a major lipid constituent of microsomes, affected DNP mutagenicity similarly. An increase in PC concentration resulted in a greater inhibitory effect. The reduction in DNP mutagenicity observed with microsomal lipids or with PC was less than that detected with uninduced S9, whilst the mutagenicity of 2-nitrofluorene was reduced to an approximately equal extent by lipids and S9. This phenomenon may be responsible for the S9-mediated detoxification of other mutagenic nitroaromatic compounds and may have important implications for mutagenicity testing.

Distribution and excretion of 1,8-dinitro[144C]pyrene in the mouse

Abstract The disposition of the environmental pollutant and potent mutagen and carcinogen, 1,8-dinitropyrene (DNP) in female BALB/c mice was investigated. In the first 48 h after oral administration of 1,8-dinitro [4,5,9,10-14C]pyrene ([14C]DNP), 42% of the dose was eliminated in the faeces and 12% in the urine. Faeces was the major pathway of excretion with 45% of the dose being eliminated by this route in 9 days. Distribution of DNP in various tissues (blood, liver, spleen, lungs, kidneys, stomach, small and large intestine was studied over 9 days. There was a linear increase in the concentration of radioactive material in the blood, liver and kidneys up to 6 h after [14C]DNP administration, representing 0.27, 2.9 and 0.21% of the radioactive dose, respectively. The corresponding figures after 24 h decreased to 0.1, 1.6 and 0.12%, respectively. In comparison, radioactivity present in the spleen and lungs was low and did not significantly change with time. In studies with ligated sections of the gastrointestinal tract, DNP absorption was from the small and large intestine and there was none from the stomach. The rate of absorption of DNP from the small intestine was greater than that from the large intestine, although overall uptake of the compound was poor (more than 80% of the original dose was recovered from the ligated small intestine after 120 min). The data from these studies suggest that although absorption of orally administered DNP is slow, the compound or its metabolites persist in the body for long periods and the liver should be considered as the major target organ.

Inhibition of dinitropyrene mutagenicity in vitro and in vivo using Salmonella typhimurium and the intrasanguinous host-mediated assay

Dinitropyrenes (DNP), present in polluted air, are potent direct-acting mutagens in Salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of rat-liver S9 or microsomes. This has now been confirmed using mouse hepatic fractions. Since most in vitro test systems do not adequately simulate conditions encountered in the intact animal, we have investigated dinitropyrene mutagenicity to Salmonella in the host-mediated assay. 1,8-Dinitropyrene (1,8-DNP) given p.o. to BALB/c mice induced a weak mutagenic effect in S. typhimurium TA98 recovered from the liver 1 h after i.v. administration (optimum time). Over the entire dose range tested no toxicity to bacterial cells was detected. Mutation induction in vivo was dose-related with maximum response at 1 mg DNP/kg body weight. This optimum dose, however, was non-mutagenic to strains TA98/1,8-DNP6 (O-transacetylase-deficient) or TA98NR/1,8-DNP6 (nitroreductase- and O-transacetylase-deficient). 1,3-Dinitropyrene and 1,6-dinitropyrene were weakly mutagenic to TA98 at doses similar to 1,8-DNP. Studies with [14C]1,8-DNP showed that 1 h after oral dosing (1 mg/kg), over 100 ng of 1,8-DNP equivalents were present in the liver (= 0.73% dose). However, only about 5.5 ng were present in the bacterial pellet, suggesting that hepatic components in vivo, as in vitro, bind to DNP, thus interfering with its interaction with Salmonella.