Biomedical subjects
Publications and source records attributed to W Klinger.
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Several subpopulations of hepatocytes with increasing cell diameters were isolated, the smaller cells were attributed to the periportal area, the larger ones to the perivenous region. Profiles of total cytochrome P-450, benzphetamine N-demethylation and ethoxyresorufin O-deethylation, cytochrome c-reductase, glucose-6-phosphatase and GPT activities were determined. With adult hepatocytes an increasing cytochrome P-450 concentration with increasing cell diameter could be observed, paralleled by increasing activities of monooxygenases. Glucose-6-phosphatase and GPT also revealed increasing activities with increasing cell diameter, but cytochrome c-reductase did not show a distinct zonation. Immature hepatocytes (age 11-15 days) were smaller, more fragile, and could not be isolated with the same enzyme solution as adult hepatocytes. They did not show any zonation of cytochrome P-450 whereas the zonation of the monooxygenases was almost fully developed. For cytochrome c-reductase a zonation with higher activities in the perivenous cells could be demonstrated, in contrast to the lack of zonation in adult rats. Glucose-6-phosphatase showed a decline with increasing cell diameter in immature hepatocytes, whereas GPT did not show any zonation. In rats aged 20 days the zonation of these parameters in liver was in between younger and older animals.
A single intraperitoneal administration of 3-methylcholanthrene (3MC) or beta-naphthoflavone (BNF) increased aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin 0-deethylase (EROD) activities. Following a second administration of both inducers 3-4 weeks later, the extent of induction was considerably greater. Such a "booster"-phenomenon was also observed with different cytochrome P-450 (P-450)-dependent N-demethylation reactions after repeated phenobarbital (PB) administrations. Again the P-450 content was higher than after a single PB administration.
The effect of temperature on the rates of ethylmorphine N-demethylation and ethoxycoumarin O-deethylation was studied with liver homogenates prepared from 1- to 120-day-old rats. Breaks at temperatures of 21.5-28.0 degrees C were observed in the Arrhenius plots of these reactions, differences being dependent mainly on age of the animals rather than on reaction type. No breaks could be demonstrated for ethylmorphine N-demethylation with liver homogenates of 10- and 120-day-old rats and for ethoxycoumarin O-deethylation in newborn and 120-day-old rats. In poikilothermic 3-, 10- and homothermic 30-day-old rats ethylmorphine N-demethylation rate was dependent on incubation temperature (7, 20 and 37 degrees C). The relative increase in N-demethylation rate after phenobarbital treatment was most marked in 30-day-old rats at an incubation temperature of 37 degrees C. In infantile rats the relative induction was highest at 7 degrees C.
After a 3-day treatment with T3 the ethylmorphine N-demethylation rate was diminished in 10- to 60-day-old rats, whereas the ethoxycoumarin O-deethylation rate was distinctly enhanced in some age-groups, particularly in 33-day-old rats. P-450 concentration was decreased in young rats and not changed in 60-day-old ones. Phenobarbital administration enhanced the rate of both reactions and P-450 concentration in all age-groups. This treatment significantly attenuated the T3 effects on P-450 concentration and ethylmorphine N-demethylation. beta-Naphthoflavone induced ethoxycoumarin O-deethylation and P-450 concentration in all age-groups. Compared with non-induced animals, the T3 effects on ethoxycoumarin O-deethylation (increase) and ethylmorphine N-demethylation (decrease) were more distinct, whereas P-450 concentration was scarcely influenced by T3. Age-differences in T3 actions were generally diminished by inducers.
Cyclobarbital inhibited diazepam metabolism by rat liver supernatant only at concentrations of 1 mM. Ethylmorphine N-demethylation is strongly inhibited by diazepam in a non-competitive manner whereas cyclobarbital shows a competitive inhibition type. Both cyclobarbital and oxazepam are weak inhibitors. Hexobarbital- evokes typical type I spectral changes with rat liver microsomes whereas cyclobarbital and diazepam-induced spectral changes are similar to type II or inverse type I. Barbital evoked the same type of spectral changes as diazepam and cyclobarbital. With the usual concentration in vivo the metabolism of diazepam and other drugs can scarcely be influenced by cyclobarbital.
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Neither destruction of thymus by N-methylnitrosourea or by X-rays nor thymectomy or splenectomy in rats of different ages affected hexobarbital sleeping time, ethylmorphine N-demethylation or ethoxycoumarin O-deethylation significantly and systematically. Thymectomy or thymus destruction by x-rays of newborn rats did not significantly influence postnatal development or inducibility by phenobarbital of the monooxygenase reactions.
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The influence of T3 on some cytochrome P-450-dependent biotransformation reactions (ethylmorphine N-demethylation, ethoxycoumarin O-deethylation and ethoxyresorufin O-deethylation) was investigated in rats of different ages. After T3 administrations on 3 consecutive days to rats of different ages, on the 4th day ethylmorphine N-demethylation rate was diminished in all age groups. On the contrary, ethoxyresorufin and ethoxycoumarin O-deethylation rates were considerably enhanced, preferentially in 33-day-old animals. The P-450 concentration was increased to a smaller degree. After T3 treatment on the first 8 days of life long-term effects on ethylmorphine N-demethylation were observed. Low T3 doses accelerated this reaction in 33- and 60-day-old rats. Ethoxycoumarin O-deethylation was not influenced. T3 administrations cannot diminish age-differences in drug metabolism.
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The in vitro influence of CdSO4 on the liver microsomal benzphetamine-N-demethylation and ethoxyresorufin-O-deethylation activities in male Wistar rats/age 10, 30, 60 and 240 days/as well as the type of inhibition were studied after phenobarbital or beta-naphthoflavone pretreatment. Both reactions were inhibited by CdSO4 in dependence on concentration in all age groups. After phenobarbital pretreatment the inhibition of N-demethylation was slightly pronounced in 10-day old rats. In 10 and 30-days old rats the type of inhibition was competitive while in 60 and 240-days old rats a non-competitive type of inhibition was observed. O-deethylation of ethoxyresorufin was inhibited also in 10-day old rats, the inhibition type was non-competitive in all age groups. After beta-naphthoflavone pretreatment N-demethylation was inhibited competitively in all age groups. The inhibition of O-deethylation was more pronounced in 30-day old rats, in other age groups the inhibition was weak but concentration dependent. The type of inhibition was noncompetitive for all age groups studied.
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The experiments were designed to investigate some details of the action of 3-methylcholanthrene (3-MC) on the regulation of transcription. After a single intraperitoneal dose of 3-MC a significant increase in the activities of both nucleolar and nucleoplasmic protein kinases in hepatic cells of young rats was found. The maximal stimulation took place 24 hr after the administration of 3-MC and the extent of activation was much greater in the nucleolar fraction. There is a significant elevation of the activities of both functional forms, free and template-engaged, of RNA polymerase A 24 hr after a single injection of 3-MC. Free and engaged forms of extranucleolar RNA polymerase B show a different behaviour: after 24 hr of 3-MC administration the engaged form is markedly enhanced while the activity of the free enzyme shows a significant decrease. The more moderate increase in total RNA polymerase B activity is obviously preceded by a transfer of the enzyme from 'free' to 'engaged' form. Since the enhancement of protein kinase activities was accompanied by the stimulation of nuclear RNA polymerases we suggest that both kinds of enzymes are involved in an epigenetic mechanism of the inducing action of 3-MC on cytochrome P1-450.
The widely applied Nash-method and its modifications have unsatisfactory specificity and restricted sensitivity for the determination of small amounts of formaldehyde. A method using small volumes of reaction mixtures with low background absorbance is described; this method avoids dilution during the protein denaturation step by the use of trichloroacetic acid. Alternatively a 'tryptophan-sulfuric acid-iron' reaction takes advantage of high specificity for formaldehyde detection. A more economical and sensitive variant is described including trichloroacetic acid precipitation of protein. The sensitivity and specificity of the colour development reaction of the 'tryptophan-sulfuric acid-iron' reaction appears to be superior to the Nash-modification. However the detection of formaldehyde by the NASH method can also be amplified by subsequent extraction and concentration of the reaction product diacetyl-dihydrolutidine into n-amyl alcohol so that a formaldehyde amount of few nanomoles can be determined. The application of these methods is recommended in cases of formaldehyde formation in small amounts such as in cell cultures or when kinetic data at low substrate concentrations and slow turnover rates have to be measured.
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Cadmium++ added in vitro destroys rat liver cytochrome P-450 (cyt. P-450) with increasing age by 25-50%. Ethylmorphine N-demethylation is inhibited only in rats 30-days old and thereafter. Ethoxycoumarin 0-deethylation is inhibited even in newborn rats, and the maximal inhibition appears to increase with age. It is concluded that in all age groups cadmium resistant cyt. P-450 subspecies are present. Ethoxycoumarin 0-deethylase activity possibly indicates the cadmium sensitive P-450 fraction.