Metabolizing activities of postmitochondrial liver fractions (S-9) on benzo[a]pyrene and dimethylnitrosamine.

The mutagenic activity of the carcinogens benz[a]pyrene and dimethylinitrosamine after being metabolised by postmitochondrial liver fractions (S-9) from rats, Syrian hamsters and rabbits was estimated in the Ames' test. The liver donors were pretreated with phenobarbital, 3-methylcholanthrene, PCB (Delor 106) or peanut oil. Parallel to the mutagenicity tests, enzymes of S-9 such as dimethylnitrosamine demethylase and aryl hydrocarbon hydroxylase were estimated and their activity evaluated with reference to the results of the Ames' test.

The role of metabolism of chemicals with respect to carcinogenicity tests.

Precarcinogens can be activated to carcinogenic effective compounds--ultimate carcinogens--by mammalian enzymes in vivo. In in vitro short-term tests, estimating effects which correlate with carcinogenicity in vivo of a chemical, these enzymes are represented by liver postmitochondrial fractions (S9) added to the test system. For the characterization and standardization of these S9 preparations we estimated certain enzymes involved in activating process: benzo(a)pyrene hydroxylase, the N-demethylases of dimethylnitrosamine, 4-dimethylaminoazobenzene and for a comparison, ethylmorphine as well as the azoreductase with 4-dimethylaminoazobenzene as a substrate. In order to get preparations with high enzyme activities, the animals such as rats, mice, hamsters and rabbits were pretreated by inducers such as polychlorinated biphenyls, phenobarbital, and 3-methylcholanthrene. We have observed species-specific differences in basic enzymatic patterns and in the degree of enzyme activities after induction. So it is principally possible to find out, whether or not a distinct animal species or strain is capable of metabolizing certain precarcinogens. Our aim is to optimize the evaluation of the carcinogenic risk of chemicals to humans by including human enzyme preparation in short-term tests.

Comparison of spectral properties of 3-MC induced cytochrome P-448 from rabbits and rats.

EPR and optical spectral properties of cytochrome P-488 from 3-methylcholanthrene-induced rabbits are compared with those of rats. In the EPR spectra at 77K and in the optical absorption spectra at room temperature a considerable temperature independent high spin content of the rabbit cytochrome is observed which has been estimated to about 55% by titration with n-octylamine. On the other hand the high spin content of the rat cytochrome depends strongly on temperature and amounts to about 6% at 5 degrees C and to about 35% at 34 degrees C. The binding of substrates and ligands to the rabbit cytochrome as well as its reduction by sodium dithionite are slower as compared with the rat cytochrome but also with phenobarbital-induced cytochrome P-450 from rats and rabbits. Contrary to the 3-methylcholanthrene induced cytochrome P-448 from rats, that from rabbits does not bind 3-methylcholanthrene. A particular protein structure establishing the high spin state and an absent binding site for type I substrates is assumed to be responsible for these and other peculiarities of cytochrome P-448 from 3-methylcholanthrene-induced rabbits.

Comparison of microsomal and solubilized monooxygenases from rat and rabbit by proton magnetic relaxation.

The paper presents results of a comparative study of the haem environment, by proton magnetic relaxation, in P-450 and P-448 monooxygenases from rat and rabbit, induced by phenobarbital and 3-methylcholanthrene, in both species. It was established that the method yields information on the accessibility of the haem iron for solvent molecules (protons), both in microsomes and in solubilized samples of various degrees of purification, i.e. association. The state of micelles in the solutions does not alter the haem iron accessibility. A slight difference was found for the microsomes suspended in a phosphate vs. pyrophosphate buffer, but this is without any consequence with regard to the species and form differences. The correlation time for the highly purified LM2 fraction of rabbit P-450 could not be determined more precisely than before for a sample of lower purity, because the relaxation rates are frequency independent. The correlation time for the rat P-448 monooxygenase was determined by dispersion measurements to be (4.1 +/- 0.4) x 10(-11) s. It was found that the PMRx behaviours of rabbit and rat monooxygenases are more alike in microsomes than in the partially purified solubilized form. The solubilization produces a pronounced alteration of the PMRx temperature dependence only for the rat 3-MC induced monooxygenase P-448. For the P-450 form the haem iron becomes less accessible on solubilization, both for the rabbit and the rat liver monooxygenases, whereas in case of rat liver P-448 the accessibility is considerably enhanced on solubilization. There is a substantial structural specificity of the haem environments from the two animal species, the one from rat being tighter. The reduced, NO-bound rabbit liver monooxygenase was studied also, but the results are not yet conclusive, except the fact that the unpaired spin from NO is thoroughly shielded from the solvent compared with the haem iron from the original sample. The following series of increased haem-iron accessibility emerges from the PMRx studies known so far: rat (P-448) less than rabbit (P-448) less than rat (P-450) less than rabbit (P-450) in microsomes, and rabbit (P-448, with 3-MC bound?) less than Pseudomonas putida (P-450) rat less than (P-448), less than rat (P-450) less than rabbit (P-450) from solubilized samples. For the latter, it appears that increased enzymic specificity goes along with a closing of the haem cleft.

[Free fatty acids as inhibitors of the respiratory chain in testicular homogenates]. / Unveresterte Fettsäuren als Hemmstoffe der Atmungskette in Hodenhomogenaten.

The supernatants of the 440 000 . g . min centrifugation of homogenates of rat, bull and boar testicles and sperm inhibit the NADH-oxidase activity of non-phosphorylating submitochondrial particles (ETP). Whereas no inhibitory activity was observed with young rats (150 g), a marked inhibition was detected with heavier animals. The inhibitory activity of testicles was located in the microsomal fraction. The reaction of the testicular inhibitor with the ETP is initiated by an instant reversible binding followed by a slow irreversible inhibition of the electron transport. The reason of the time-dependence is neither an interaction between the enzymes of the ETP and those of the microsomal electron transport nor a slow degradation of the ETP by microsomal phospholipases. Some observations indicate an indirect involvement of phospholipase via the formation of free fatty acids (FFA). The inhibitory fraction could be solubilized from the microsomes both by sodium cholate treatment and by ethanol extraction. After separation of the lipid classes by chromatography on silica gel and gas-chromatographic analysis the inhibitory fraction was identified as a mixture of free fatty acids (FFA) of different chain lengths and degree of saturation. Thus a new effect of FFA on the mitochondrial electron transport has been detected which is different from other actions known up till now. The degradation of the phospholipids of the endoplasmic reticulum in the spermatozoa may be the source of the enhanced formation of FFA. An inhibition of the cell respiration presumably does not occur in vivo. The high FFA level in the testicular homogenates of sexually mature animals is a consequence of an intensive FFA metabolism, especially of high phospholipase activity.

[Induction of tumors in Wistar-rats after oral application of aminopyrine and nitrite (author's transl)]. / Induktion von Tumoren nach oraler Applikation von Aminophenazon und Nitrit bei Wistar-Ratten.

In a long-term experiment the induction of tumors in rats after oral application of aminopyrine and nitrite by gastric intubation was investigated. The animals treated with aminopyrine and nitrite (test-group) had an incidence of different types of liver tumors of 96% in females and 97% in males. The percentage of liver tumors was increased significantly in the test-group in comparison to all control-groups. Besides the tumors of the liver other types of tumors were stated to a minor degree, but there was no difference between the test-group and the control-groups.

Infrared spectral studies of carbon monoxide complexes of microsomal cytochromes P-450 and P-448.

The characteristic infrared spectral parameters of the carbonyl stretching vibration (the maximum position nu CO, the band width at half height delta nu 1/2 and the apparent molar extinction coefficient epsilon M) have been determined for the CO complexes of microsomal cytochromes P-450 (PB-induced) and P-448 (3-MC-induced) from rabbits and rats. The cytochromes P-450 and P-448 from the same species as well as the cytochromes prepared by the same inducer but from the two different species yielded nu CO band parameters different from each other and from the bacterial cytochrome P-450cam [1]. These differences are discussed in connection with (1) the presence of different protein moities (multiple forms) of cytochromes, (2) changes in the order of the nearest neighbour environment (accessibility) of the CO binding site and (3) the presence of (endogenous bound substrate. The heme-carbonyl of the microsomal cytochromes senses subtle changes in the nearest heme environment by changing the solvent from H2O to D2O.

[Action of polychlorinated biphenyls (Kanechlor 500 and Delor 106) and 3-methylcholanthrene on the activity of some rat liver microsomal enzymes]. / Die Wirkung von polychlorierten Biphenylen (Kanechlor 500 und Delor 106) und von 3-Methylcholanthren auf die Aktivität mikrosomaler Enzyme der Rattenleber.

The influence of 3-methylcholanthrene and two commercially available mixtures of polychlorinated biphenyls (Kanechlor 500 and Delor 106) on the activity of some microsomal rat liver enzymes has been investigated. The studies included the demethylation of ethylmorphine, dimethylnitrosamine, and 4-dimethylaminoazobenzene as well as the investigation of the activity of aryl hydrocarbon hydroxylase and of azoreductase using benzo(a)pyrene and 4-dimethylaminoazobenzene as substrates, respectively. 3-methylcholanthrene induced the aryl hydrocarbon hydroxylase and azoreductase and led to an increase in the demethylation of 4-dimethylaminoazobenzene but not in the ethylmorphine demethylation. The relatively low increase in the dimethylnitrosamine demethylation was not statistically significant. These polychlorinated biphenyls caused a significant increase in all the enzyme activities studied. In most cases Delor 106 was more active than Kanechlor 500. The results are discussed and compared with those of other authors.

Effects of some inhibitors on the nitrosation of drugs in human gastric juice.

The nitrosation of several drugs under simulated stomach conditions was only weakly inhibited by the beverages studied. With easily and rapidly nitrosatable drugs, such as aminophenazone or piperazine, administered orally, the use of an inhibitor of nitrosation is to be recommended. Of all the substances investigated, ascorbic acid should be regarded as the best inhibitor because of its pronounced activity at the pH values occurring in the stomach and its lack of toxic effects. We would like to propose that the drugs under consideration should be made up to contain a sufficient amount of ascorbic acid.