Validation of a polymerase chain reaction method for the detection of rendered bovine-derived materials in feedstuffs.

This study validated a polymerase chain reaction-based method for the detection of a specific bovine mitochondrial gene derived from rendered bovine tissues and admixed with complete animal feed. Four laboratories participated in this effort: one state laboratory and three Food and Drug Administration (FDA) laboratories, including one FDA field laboratory. The protocol used a statistical approach of 90% probability, with a 95% confidence interval for determining acceptable rates of false-positive and false-negative samples. Each participating laboratory analyzed 30 samples of feed each containing 0, 0.125, and 2.0% bovine meat and bone meal (BMBM), for a total of 90 feed samples. The samples were randomized such that the analysts were unaware of the true identity of the test samples. The results demonstrated that all laboratories met the acceptance criteria established for this protocol. The overall rates of false-negative results were 0.83% (1/120) at the level of 0.125% BMBM and 1.67% (2/120) at the level of 2% BMBM. The overall rate of false-negative results for all levels of BMBM was 1.25% (3/240). The rate for false-positive results was 0.83%.

Host-mediated mutagenicity experiments with benzo[a]pyrene and two of its metabolites.

Benzo[a]pyrene (BP) and two of its major metabolites, the ultimate mutagen BP-4,5-oxide and the proximate mutagen trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol) were investigated for mutagenicity in Salmonella typhimurium TA1538, TA98 and TA100 using an intrasanguineous host-mediated assay. BP and BP-4,5-oxide were not mutagenic under any experimental conditions. BP-7,8-diol was inactive with the strain TA1538 but was mutagenic with the strains TA98 and TA100. The effect was potentiated by pretreatment of the host mice with the cytochrome P-450 inducer 5,6-benzoflavone. We conclude: (i) one of the reasons for the observed insensitivity of the intrasanguineous host-mediated assay towards BP is that BP-4,5-oxide, which contributes to the microsome-mediated mutagenicity of BP, is inactive in the host-mediated assay; (ii) the finding that BP-7,8-diol is mutagenic in the host-mediated assay demonstrates that the lack of mutagenicity of BP is not intrinsic; (iii) the potentiated mutagenicity after treatment of the hosts with 5,6-benzoflavone suggests that cytochrome P-450 is more important in the activation of BP-7,8-diol in this system than other enzymes (e.g. prostaglandin synthase) that can also activate this compound in vitro.

The mutagenicity of dibenz [a,h]anthracene activated by phenobarbital-inducible mouse-liver mono-oxygenase is potentiated by the presence of hydrophilic residues at the K-region of the molecule.

Dibenz[a,h]anthracene and synthetic K-region derivatives of the parent hydrocarbon and of benz[a]anthracene were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA98, TA100 and TA1537. The K-region metabolite 5,6-dihydroxy-5,6-dihydrodibenz[a,h]anthracene, inactive as such, was efficiently activated to mutagens for TA98 and TA100 by mouse-liver 9000 X g supernatant or microsomal fraction. Microsomes from phenobarbital- or Aroclor-1254-treated mice were efficient for this activation, while those from untreated or beta-naphthoflavone-treated mice were much less active. A study on the influence of various structural features on this efficient activation by phenobarbital-inducible mono-oxygenase of mouse-liver microsomes showed that, if the K-region were saturated, no metabolism to mutagens occurred, while substitution of the K-region by carbonyl and hydroxyl substituents led to increased mutagenic efficacy with increasing hydrophilicity (dihydro less than carbonyl less than hydroxyl). The K-region epoxide was the only derivative that did not require metabolic activation and it had a markedly different mutagenic specificity in that it was also mutagenic for TA1537.

Activation of phenanthrene to mutagenic metabolites and evidence for at least two different activation pathways.

Phenanthrene, generally considered to be a non-carcinogen, was converted by mammalian tissue preparations to products that were mutagenic for Salmonella typhimurium TA100 and TA1537. In TA100 the mutagenic response was highly dependent on the activation system used. High amounts of 9000 x g supernatant fraction from the liver of rats induced by Aroclor 1254 were required. Equivalent amounts of microsomal or cytosolic fraction alone did not activate phenanthrene to an observable extent. Furthermore, this activation was only observed when the rats had been treated with Aroclor. Liver preparations from control rats and from rats treated with phenobarbital, beta-naphthoflavone, a mixture of both, and transstilbene oxide failed to activate phenanthrene to mutagens for TA100. Interestingly, liver microsomes and 9000 x g supernatant fractions of Aroclor-treated mice also failed significantly to activate phenanthrene to mutagens for this strain. Addition of pure epoxide hydrolase to the S9 mix had no influence on this activation. Glutathione (GSH) decreased the mutagenicity, but uridine diphosphate glucuronic acid (UDPGA) had only minor effects. An adenosine-3'-phosphate-5'-sulfate phosphate (PAPS) generating system, however, increased the number of his+ revertants from TA100 (2.7-fold). TA1537 was reverted by mutagens produced from phenanthrene by liver microsomes or 9000 x g supernatant fraction, when the microsomal epoxide hydrolase was inhibited by 1,1,1-trichloropropene oxide. This activation pathway exists in Aroclor-treated rats and mice. The results show that at least 2 different pathways for metabolic activation of phenanthrene exist which were observed in 2 differentially sensitive tester strains and distinguished by their different metabolic requirements. Furthermore, the study shows that earlier suggestions do not hold that equivalent results can be obtained by inducing animals with a combination of phenobarbital and beta-naphthoflavone instead of the environmentally persistent Aroclor 1254. Moreover, the study provides a striking example that the use of 9000 x g supernatant in amounts corresponding to standard practice but sub-optimal for a particular compound only impede the detection of a weak mutagen and that the rapid inactivation of active metabolites by inactivating enzymes may be responsible for negative results in mutagenicity testing.

Mutagenicity of phenanthrene and phenanthrene K-region derivatives.

Phenanthrene and 9 K-region derivatives, most of them potential metabolites of phenanthrene, were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 and the rec assay with Bacillus subtilis H17 and M45. The strongest mutagenic effects in the reversion assay were observed with phenanthrene 9,10-oxide, 9-hydroxyphenanthrene and N-benzyl-phenanthrene-9,10-imine. Interestingly, the mutagenic potency of the arene imine was similar to that of the corresponding arene oxide. This is the first report on the mutagenicity of arene imine. The mutagenic effects of all these phenanthrene derivatives were much weaker than that of the positive control benzo[a]pyrene 4,5-oxide. Even weaker mutagenicty was found with cis-9,10-dihydroxy-9,10-dihydrophenanthrene and with trans-9,10-dihydroxy-9-10-dihydrophenanthrene. The other derivatives were inactive in this test. However, 9-10-dihydroxyphenanthrene and 9,10-phenanthrenequinone were more toxic to the rec- B. subtilis M45 strain than to the rec+ H17 strain. This was also true for phenanthrene 9,10-oxide and 9-hydroxyphenanthrene, but not with the other test compounds that reverted (9,10-dihydroxy-9,10-dihydrophenanthrenes; N-benzyl-phenanthrene 9,10-imine; benzo[a]pyrene 4,5-oxide) or did not revert (phenanthrene, 9,10-bis-(p-chlorophenyl)-phenanthrene 9,10-oxide, 9-10-diacetoxyphenanthrene) the Salmonella tester strains. Although the K region is a main site of metabolism and although all potential K-region metabolites were mutagenic, phenanthrene did not show a mutagenic effect in the presence of mouse-liver microsomes and an NADPH-generating system under standard conditions. However, uhen epoxide hydratase was inhibited, phenanthrene was activated to a mutagen that reverted his- S. typhimurium. This shows that demonstration of the mutagenic activity of metabolites together with the knowledge that a major metabolic route proceeds via these metabolites dose not automatically imply a mutagenic hazard of the mother compound, because the metabolites in question may not accumulate in sufficient quantities and therefore the presence and relative activities of enzymes that control the mutagenically active metabolites are crucial. N-Benzyl-phenanthrene 9.10-imine was mutagenic for the episome-containing S. typhimurium TA98 and TA100 but not for the precursor strains TA1538 and TA1535. This arene imine would therefore be useful as a positive control during routine testing to monitor in the former strains the presence of the episome which is rather easily lost.