Joseph E. Sinsheimer

Mutagenicity of aliphatic epoxides

The mutagenicity of 17 aliphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains TA100 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most potent mutagens in both strains. 1,1-Disubstitution resulted in the complete loss or reduction of mutagenicity, trans-1,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-1,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the presence of electron-withdrawing substituents increased mutagenicity.

Comparison of the adrenalytic activity of mitotane and a methylated homolog on normal adrenal cortex and adrenal cortical carcinoma

SummaryMitotane is an important adrenalytic drug for the treatment of adrenal cancer whose use is limited by toxicity. Reports from another laboratory indicated that a methylated homolog of Mitotane (Mitometh) tested in guinea pigs possessed comparable adrenalytic activity but was less toxic than Mitotane. This observation prompted us to undertake a comparative study of these two drugs on the basis that Mitometh may be a superior agent for the treatment of adrenal cancer. Preliminary studies in guinea pigs failed to show a significant adrenalytic effect for either Mitotane or Mitometh. Thus, we extended the study to 13 mongrel dogs weighing 12–15 kg that were treated daily with Mitometh or Mitotane (50–100 mg/kg) for 6 or 12 days. Cortisol decreased to undetectable levels and adrenocorticotropic hormone (ACTH) rose to 10 times the baseline levels within 72 h in Mitotane-treated animals. Despite the achievement of similar drug levels, Mitometh treatment in dogs failed to suppress cortisol or increase ACTH. To determine whether these differences were due to differences in bioavailability, we measured the relative concentration of Mitotane and Mitometh in homogenates of adrenal cortex obtained from Mitotane- and Mitometh-treated dogs. The adrenal concentration of Mitometh determined in Mitometh-treated dogs was 5 times higher than the concentration of Mitotane measured in Mitotane-treated animals. Whereas the adrenal glands of Mitotane-treated dogs showed hemorrhage and necrosis, the Mitometh-treated animals showed no adrenal damage. Despite the lack of adrenalytic activity, Mitometh maintained its toxicity as demonstrated by microscopic evidence of hepatic necrosis and an increase in hepatic enzymes. The adrenalytic effects of both agents was also studied in vitro using a human functioning adrenal cortical carcinoma cell line. NCI-H295. Whereas Mitotane strongly suppressed cell growth, Mitometh had a weaker effect. We conclude that Mitometh is not likely to be effective in the therapy of adrenal cancer. Moreover, the results of this study are supportive of the view that metabolic transformation of Mitotane is in some way linked to its adrenalytic action.

Sister-chromatid exchange and chromosome aberrations for 4 aliphatic epoxides in mice

Sister-chromatid exchange (SCE) and chromosome aberrations (CA) in bone marrow cells were analyzed after in vivo exposure in mice to 4 aliphatic epoxides, namely 1-naphthyl glycidyl ether (NGE), 1-naphthyl propylene oxide (NPO), 4-nitrophenyl glycidyl ether (NPGE) and trichloropropylene oxide (TCPO). These compounds were selected as being among the most mutagenic aliphatic epoxides in our previous structure-mutagenicity studies with the Ames test. There were significant dose-related increases in SCE and CA results for all 4 epoxides. The order of genotoxicity as established through SCE was NGE greater than NPO greater than NPGE approximately equal to TCPO greater than solvent control. It is of interest that Ames Salmonella results are consistent with in vivo genotoxicity for these compounds. However, only the plate test version of the Ames procedure is consistent with this order of in vivo genotoxicity and neither preincubation Ames testing results nor chemical alkylation rates would have predicted this order.

The genotoxicity of enantiomeric aliphatic epoxides

The (R)- and (S)-optical isomers of 9 epoxides, benzyloxymethyloxirane, epichlorohydrin, glycidol, glycidyl 3-nitrobenzenesulfonate, glycidyl 4-nitrobenzoate, glycidyl tosylate, styrene oxide, glycidyl 1-naphthyl ether and glycidyl 4-nitrophenyl ether, have been compared for their in vivo and in vitro genotoxicity. The in vitro short-term test employed was the Ames mutagenicity assay with Salmonella strain TA100. The in vivo tests were chromosomal aberrations (CA) as well as sister-chromatid exchange (SCE) in bone-marrow cells of mice following intraperitoneal administration of these epoxides. Differences in mutagenicity between isomers were established with TA100 for all the compounds. While 13 of the isomers were genotoxic compared to a negative control by CA measurements, only in the case of glycidyl 4-nitrobenzoate could a significant difference be found between isomers by this test. However, with SCE evaluations, differences were detected between the (R)- and (S)-isomers for all the pairs of compounds with the exception of those for benzyloxymethyloxirane and glycidyl 4-nitrophenyl ether. At least in part, differences in the patterns of genotoxicity among compounds can be related to their differences in reaction pathways.

A Sensitive Fluorimetric Procedure for the Determination of Aliphatic Epoxides under Physiological Conditions

A highly sensitive fluorimetric procedure based on alkylation of nicotinamide is described for the determination of aliphatic epoxides. Subsequent reaction of the resulting N-alkylnicotinamides with a ketone in basic medium yields strongly fluorescent products after final acidification. Sensitivity of the assay is in the picomole range with good reproducibility. The alkylation reaction proceeds under physiological conditions and thus shows potential for the analysis of epoxides in biological materials. Despite rapid enzymatic detoxification, styrene oxide could be directly detected in 9000g supernatant liver fractions using the present approach. Aliphatic epoxides have been demonstrated to be intermediates in the biotransformation of several ethylenic compounds (l-4). Their high chemical reactivity accounts for such biological effects as mutagenesis (57), binding to cellular macromolecules (g-IO), and alkylation of nucleic acids (11). Some epoxides are widely used in industry and thus represent potential hazards for exposed workers. So far, detection methods are restricted to gas-liquid chromatography (gc)’ (14) or a calorimetric p-nitrobenzylpyridine (NBP) approach (7,12,13). The gc method, while offering the advantage of specificity, requires extraction when it is applied to biological materials, which might result in substantial degradation. Its sensitivity is generally limited to the nanomole range. The NBP method has improved sensitivity but with the disadvantage of instability of its final color. In addition, the reaction can only ’ Present address: Laboratoria voor Medische Biochemie en voor Klinische Analyse, R.V.G., DePintelaan 135, B-9000 Gent, Belgium. * To whom correspondence should be addressed.

BACTERIAL MUTAGENICITY AND TOXICITY OF CYCLOALIPHATIC EPOXIDES

The mutagenicity of 12 cycloaliphatic epoxides was investigated using the Ames Salmonella assay without the addition of liver homogenate fractions. Base-pair substitution mutagenic activity was detected for 8 members of this series of compounds, confirming our laboratorys previous observations of weak mutagenic response at high dose levels for cis-1,2-disubstituted epoxides. While mutagenicity decreased with expanding ring size, inhibition of bacterial growth increased for increases in ring size. Toxicity accompanying the required high doses for the demonstration of mutagenicity for these compounds prevented the establishment of meaningful dose-response ranges for the remaining epoxides tested. The volatility observed with these oxiranes also made dose-response establishment difficult but was countered by the use of petri dish sealing bands during incubation. Mutagenicity in this series was found to be generally more pronounced in TA1535 while toxicity was detected with greater sensitivity by TA100. The use of the less permeable strains TA92, TA1950 and TA2410, all having normal lipopolysaccharide cell wall coatings, failed to reduce this marked toxicity. The repair test compared results in TA1535 (repair-deficient strain) with TA1975 (repair-proficient strain) and demonstrated that the bacteria were not being killed by damage to DNA since toxicity was not reduced in TA1975.

Isolation and antiviral activity of the gymnemic acids

Aus den Blättern vonGymnema sylvestre wurden 4 Gymneasäuren (A, B, C und D) isoliert. Die Antivirusaktivität der Säuren A und B wurde geprüft.

Mutagenicity of para-substituted α-methylstyrene oxide derivatives with Salmonella

Abstract A series of 5 para-substituted α-methylstyrene oxide derivatives have been synthesized and together with α-methylstyrene oxide as well as styrene oxide have been studied as to their mutagenicity with the TA100 and TA1535 strains of Salmonella typhimurium. A multiple regression analysis model has been developed which describes the mutagenicity of the α-methylstyrene oxides in TA100. An increase in van der Waals volume was the most important variable in the model with greater improvement occurring with inclusion of the Hammett values for the para substituents on the compounds. The α-methylstyrene oxides were less active alkylating agents with 4-(p-nitrobenzyl)pyridine than styrene oxide and with pyridine all reactivity was at the β-epoxide carbon. However all the α-methylstyrene oxide derivatives, except for the bromo compound where toxicity was evident, showed mutagenicity values either greater or comparable to that of styrene oxide. These studies would indicate that reactivity at the β-carbon should also be a factor in describing the mutagenicity of the parent styrene oxide series.

Fluorescein isothiocyanates: Improved synthesis and purity: Spectral studies

Abstract Modification of the methods of synthesis by direct reaction of the isomeric aminofluoresceins with a limited excess of thiophosgene in the presence of calcium carbonate has yielded the 5- and 6-isothiocyanatofluoresceins of high purity. The ir and nmr spectra of these isomers are described.

Adrenal proteins bound by a reactive intermediate of mitotane

Abstract Purpose: Mitotane (o,p′-DDD), is the only adrenolytic agent available for the treatment of adrenocortical carcinoma. Previous studies have shown that mitotane covalently binds to adrenal proteins following its metabolism in adrenocortical tissue to a reactive acyl chloride intermediate. It was the objective of this study to compare the electrophoresis separation patterns of such adducts following activation of mitotane by various adrenocortical sources. Methods: With the use of a 125I-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, gel electrophoresis patterns were obtained for homogenates from bovine, canine and human adrenocortical preparations as well as from a human adrenal preparation. Western immunoblotting analysis was used to test the resulting patterns for adducts of cytochrome P-450SCC and adrenodoxin. Results: The electrophoresis separations were similar for all preparations, with bands at apparent molecular weights of 49.5 and 11.5 kDa being the most pronounced. Radiolabeling of the proteins of a human adrenal cancer cell line NCI H-295 was weak, but a band at 11.5 kDa was detected. Western immunoblotting analyses indicated that the band at 49.5 kDa corresponded in molecular weight to that of adrenal cytochrome P-450SCC, but the band at 11.5 kDa did not correspond to adrenodoxin. Conclusions: The similarity of the results with canine and bovine adrenal preparations to that of human material offers useful systems for studying mitotane and its analogs. This should aid in understanding the mechanism of action of mitotane and in the design of compounds for the treatment of adrenocortical carcinoma.