Mercury in a commercial preparation of rat amylin.

The biological activity of amylin is reported to vary widely depending on the source and purity of the material. Three commercial samples of rat amylin were compared for structural differences. The samples were nearly identical using most of the available analytical measures--amino acid analysis, HPLC retention, even Edman sequencing data. When the samples were compared by ion spray ionization mass spectrometry, the molecular mass of one sample was 200 daltons higher than anticipated. Careful analysis of the sample, including atomic emission spectrometry, revealed that a mercury atom was associated with the polypeptide. The mercury presumably resulted from a deprotection step in the synthesis, involving the removal of an acetamidomethyl group from cysteine.

Mutagenicity and tumorigenicity of dihydrodiols, diol epoxides, and other derivatives of benzo(f)quinoline and benzo(h)quinoline.

The mutagenic activities of benzo[f]quinoline, benzo[h]quinoline, and a number of their derivatives, including dihydrodiols, K-region oxides, diol epoxides, and tetrahydroepoxides, were assessed in strain TA 100 of Salmonella typhimurium. The dihydrodiol derivatives of benzo[f]quinoline and benzo[h]quinoline were also tested for tumorigenic activity in newborn mice. Benzo[f]quinoline was metabolically activated in the presence of rat liver S-9 preparation to products mutagenic to the bacterial system to a greater extent than was benzo[h]quinoline. However, trans-7,8-dihydro-7,8-dihydroxybenzo[f]quinoline was less mutagenic compared to trans-7,8-dihydroxy-7,8-dihydrobenzo[h]quinoline in the presence of rat liver homogenate. The data on the mutagenic activity of the dihydrodiol derivatives of benzoquinolines were consistent with the intrinsic mutagenicity of the corresponding epoxide derivatives, in that the bay-region diol epoxides and tetrahydroepoxide of benzo[h]quinoline exhibited considerably higher mutagenic activities compared to those of the corresponding derivatives of benzo[f]quinoline at equivalent doses. The K-region oxides of benzo[f]quinoline and benzo[h]quinoline were significantly less mutagenic than their corresponding bay-region diol epoxide and tetrahydroepoxide derivatives. The demonstration that benzo[f]quinoline is significantly more mutagenic than trans-7,8-dihydro-7,8-dihydroxybenzo[f]quinoline, a precursor to the weakly mutagenic bay-region diol epoxide, suggests that the bay-region diol epoxide formation is not the principal pathway for the metabolic activation of benzo[f]quinoline to a mutagen. On the other hand, the isomeric benzo[h]quinoline appears to exert its mutagenic effect via the formation of its bay-region diol epoxide. These results indicate that the position of a nitrogen heteroatom in phenanthrene (the analogous carbocyclic aromatic hydrocarbon) not only has a marked effect on the mutagenic activities of the diol epoxide derivatives, but also can alter the metabolic activation pathways of the parent hydrocarbon. Benzo[f]quinoline, benzo[h]quinoline, and their dihydrodiol derivatives were not tumorigenic in newborn mice.

Metabolism and mutagenic activity of benzo[k]fluoranthene and 3-, 8- and 9-fluorobenzo[k]fluoranthene.

The metabolism of 3-, 8- and 9-fluorobenzo[k]fluoranthene (B[k]F) relative to B[k]F was investigated. The major metabolites of B[k]F formed in vitro using rat liver S-9 metabolism systems were 8,9-dihydro-8,9-dihydroxyB[k]F, the 2,3-quinone of B[k]F and 3-, 8- and 9-hydroxyB[k]F. Fluorine substitution within the structure of B[k]F substantially altered the types of metabolites formed in vitro. The most pronounced effect was observed with 9-fluoroB[k]F. In contrast to B[k]F, the 8,9-dihydro-8,9-dihydroxy-, 9-hydroxy- and 10,11-dihydro-10,11-dihydroxy derivatives were not detected as metabolites of 9-fluoroB[k]F. However, either the 2,3- or 4,5-dihydrodiol of 9-fluoroB[k]F was detected. In the case of 8-fluoroB[k]F, neither the 8- nor 11-hydroxy- derivatives were detected. The principle dihydrodiols formed from 8-fluoroB[k]F were the 10,11-dihydrodiol and either the 2,3-or 4,5-dihydrodiol. The pattern of metabolites formed with 3-fluoroB[k]F was similar to that observed with B[k]F with the exception that neither the 3- nor 4-hydroxy derivatives were formed. Mass spectral data indicated that fluoro substitution is not lost to any appreciable extent during the metabolism of 3-, 8- and 9-fluoroB[k]F. The mutagenic activity of these B[k]F fluoro derivatives along with B[k]F, 2,3-dihydro-2,3-dihydroxyB[k]F, the 2,3-quinone of B[k]F and 8,9-dihydro-8,9-dihydroxyB[k]F were evaluated in Salmonella typhimurium TA100 in the presence of rat liver S-9 metabolism systems. 3-FluoroB[k]F was more mutagenic than B[k]F, while both 8- and 9-fluoroB[k]F were less active. While the 2,3-dihydrodiol and 2,3-quinone were weakly active, the 8,9-dihydrodiol had similar mutagenic potency to B[k]F.

Identification of tumorigenic metabolites of benzo[j]fluoranthene formed in vivo in mouse skin.

The metabolism of benzo[j]fluoranthene (BjF) in vivo in mouse skin was investigated. trans-4,5-Dihydro-4,5-dihydroxybenzo[j]fluoranthene (BjF-4,5-diol) and trans-9,10-dihydro-9,10-dihydroxybenzo[j]fluoranthene (BjF-9,10-diol) have been identified as major metabolites. In addition, 4- and 10-hydroxybenzo[j]fluoranthene and benzo[j]fluoranthen-4,5-dione have been tentatively identified among the metabolites formed in vivo in mouse skin. The enantiomeric purity of the metabolic dihydrodiols of BjF as formed in vivo in mouse skin was determined. The major enantiomer of BjF-4,5-diol was present in 57-62% enantiomeric excess while that of BjF-9,10-diol was present in 66-71% enantiomeric excess. In each case the later-eluting enantiomer on chiral stationary-phase high performance liquid chromatography predominated. The tumor-initiating activity of trans-2,3-dihydro-2,3-dihydroxybenzo[j]fluoranthene (BjF-2,3-diol), BjF-4,5-diol, BjF-9,10-diol, and BjF was evaluated on the skin of female CD-1 mice. As a total initiation dose of 3 mumol/mouse BjF-4,5-diol resulted in a 100% incidence of tumor-bearing mice with 5.0 tumors/mouse. In comparison, BjF-9,10-diol elicited a 60% incidence of tumor-bearing mice with 1.7 tumors/mouse, while BjF-2,3-diol was inactive. At the same dose, BjF gave rise to a 90% incidence of tumor-bearing mice with 7.8 tumors/mouse. At a 1-mumol dose, BjF-4,5-diol induced a 78% incidence of tumor-bearing mice with 4.3 tumors/mouse while BjF gave rise to a 70% tumor incidence with 3.4 tumors/mouse while BjF gave rise to a 70% tumor incidence with 3.4 tumors/mouse. These studies indicate that while BjF-9,10-diol could contribute to the overall tumorigenic activity of BjF in mouse skin, BjF-4,5-diol is a more potent tumor initiator in the target tissue.

Identification of metabolites of benzo[j]fluoranthene formed in vitro in rat liver homogenate.

The metabolites of benzo[j]fluoranthene (BjF) as formed in vitro using the 9000 X g supernatant from Aroclor-pretreated rats have been identified. Two dihydrodiols, trans-4,5-dihydro-4,5-dihydroxyBjF and trans-9,10-dihydro-9,10-dihydroxyBjF have been identified as major metabolites by comparison of their spectral and chromatographic properties with those of pure synthetic standards. There was no evidence that any of the isomeric 2,3-dihydrodiol was formed as a metabolite of BjF under these incubation conditions. Neither of the metabolic dihydrodiols of BjF were formed with a high degree of stereoselectivity. The enantiomeric purity of the 4,5-dihydrodiol was 20% while that of the 9,10-dihydrodiol was 46%. At least four phenols were detected among the metabolites of BjF. These were identified as 3-, 4-, 6- and 10-hydroxyBjF based upon comparison of their UV spectra and HPLC retention times with those of synthetic reference standards. BjF-4,5-dione was also identified as a metabolite under these incubation conditions.

Tumor-initiating activity, mutagenicity, and metabolism of methylated anthracenes.

Specific methylated derivatives of anthracene are mutagenic in S. typhimurium and have tumor-initiating activity on mouse skin. In this study, the mutagenic activities of 1-, 2-, and 9-methylanthracene, 2,9- and 9,10-dimethylanthracene, 2,9,-10-trimethylanthracene, 2,3,9,10-tetramethylanthracene, and the photo-oxide of 9,10-dimethylanthracene were determined in S. typhimurium TA98 and TA100. The relative tumor-initiating activities of these compounds were also evaluated. These bioassays indicate that increased mutagenic potency and tumor-initiating activity are associated with the presence of a methyl substituent at both the 9- and 10- position of anthracene. Metabolism studies suggest that the biological activity of specific methylated anthracenes may be related to the formation of a simple epoxide adjacent to a peri-methyl substituent.

Studies on the mutagenicity and tumor-initiating activity of methylated fluorenes.

Several methylated analogs of fluorene were evaluated as mutagens in Salmonella typhimurium TA98 and TA100 in the presence and absence of microsomal activation. Among the methylated derivatives of fluorene assayed were 9-methylfluorene, 2-fluoro- and 2,7-difluoro-9-methylfluorene, 1,9-, 2,9-, 3,9- and 4,9-dimethylfluorene, 2,3,9-trimethylfluorene and 2,7,9-trimethylfluorene. Mutagenic activity was observed for several of these fluorene derivatives in the presence of rat liver homogenate. The data support a previous observation that a single methyl substituent in the 9-position of fluorene is associated with mutagenic activity within this series of compounds. Substitution with fluorine at both the 2- and 7-positions of 9-methylfluorene was not associated with a loss of mutagenic activity as evidenced by the similar mutagenic activity of 2,7-difluoro-9-methylfluorene and 9-methylfluorene. However, 2,7,9-trimethylfluorene was not mutagenic under these assay conditions. 9-Methylfluorene, 1,9-, 2,9-, 3,9- and 4,9-dimethylfluorene and 2,3,9-trimethylfluorene were active as mutagens in the presence of rat liver homogenate, but were inactive as tumor initiators when assayed on mouse skin.