J. Van Cantfort

CORRELATION BETWEEN TISSULAR AND DIVISION FUNCTIONS IN THE LIVER OF YOUNG RATS

The division and tissue specific functions are studied in the liver of young rats during the first post‐natal weeks. The division function is tested by the mitotic and the 3H‐thymidine labelling index, the specific tissular function by the cholesterol‐7 alpha‐hydroxylase activity. The nycthemeral rhythm is triggered simultaneously for the two functions at the 20th day of life. From this time, spontaneous or induced mitoses occur during the day (rest period) and the cholesterol‐7‐alpha‐hydroxylase during the evening (period of activity). The results are explained by the influence of the hypothalamoadrenal axis, which presents a circadian activity with a maximum in the evening.

Pharmacokinetics of a new anticancer drug, navelbine, in patients

SummaryA study was designed to investigate the fate of navelbine (NVB) and its excretion routes in two cancer patients treated with tritiated NVB (30mg/m2) by i.v. bolus injection. Plasma and red blood cell concentrations and urine and stool elimination were monitored over long periods of time. NVB plasma and urine concentrations were measured by both radioimmunoassay (RIA) and direct radioactive (RA) determination. Samples were also analyzed by high-performance liquid chromatography to evaluate the importance of NVB metabolism. Whereas the major excretion route for NVB was the stool (from 34% to 58.4% of the total dose given over 21 days), urinary excretion was low (about 21% within the same time period), corresponding mainly to that of unchanged drug. Thus, a good correlation was found between RIA and RA determinations in urine. In contrast, plasma area under the curve (AUC) values obtained after RA and RIA analysis differed markedly (AUC RIA/AUC RA=0.23–0.31), demonstrating that a significant proportion of the plasma-circulating drug was biotransformed, mainly during the last elimination phase. This could have important pharmacologic and toxicologic implications in clinical practice.

A new assay for glutathione S-transferase using [3H]-benzo(a)pyrene 4,5-oxide as substrate: Inducibility by various chemicals in different rat tissues compared to that of aryl hydrocarbon hydroxylase and epoxide hydratase

Abstract A new assay for glutathione S-transferase using [3H]-benzo (a)pyrene 4,5-oxide as a substrate is presented. Compared to other methods, this assay presents the following advantages: ( 1 ) it is easy and rapid since only one extraction step is required and it does not necessitate the usual Chromatographic separation of the substrate and products; (2) it uses an epoxide metabolically produced from a carcinogenic polycyclic aromatic hydrocarbon as a substrate; and (3) it is performed under conditions optimal with respect to both the pH and the concentration of the two substrates (glutathione and epoxide). Studies on the in vivo response of this enzyme to several chemicals show that glutathione S-transferase is remarkably constant and is only slightly influenced by various inducers. In this respect this enzyme behavior is very different from other enzymes involved in the polycyclic hydrocarbon metabolism. In effect, the aryl hydrocarbon hydroxylase appears in many organs to be very easily and strongly induced by several chemicals and epoxide hydratase is very significantly enhanced in the liver after phenobarbital and 16α-cyanopregnenolone treatment. These results constitute an additional argument in favor of a minor role of possible changes in the concentration of this enzyme in the regulation of polycyclic aromatic hydrocarbons elimination.

Circadian Synchronization of Liver Regeneration in Adult Rats: The Role Played by Adrenal Hormones

Abstract The role played by the adrenal hormones in the regulation of liver proliferation in adult rats was investigated under various experimental conditions.

Benzo[a]pyrene metabolism in rat fetal hepatocytes culture. Improved methodology and effect of substrate concentration

The different characteristics of benzo[a[pyrene (BP) metabolism in primary fetal rat liver cell culture have been investigated. We have determined the extent of the in vivo [3H]BP metabolism by measuring all of the metabolites retained in the cell and excreted into the culture medium. The extent of the conjugation as well as the nature of the conjugates was established and the pattern of these metabolites analyzed by high performance liquid chromatography (HPLC). The fetal hepatocytes very actively metabolize BP and readily excrete in the culture medium all the produced metabolites in the form of sulfate and glucuronide conjugates. The relative proportion of those compounds varies as a function of the substrate concentration added to the cell culture, the higher the BP concentration, the more glucuronide conjugates. The HPLC analysis of the metabolites shows that BP-1,6-quinone and -3,6-quinone are the major excreted products, indicating the probable existence of an active 6 hydroxylation reaction in the fetal hepatocytes. On the other hand, the pattern of the different metabolites is influenced by the BP concentration. At low BP doses (0.8 microM), the relative amount of polar metabolites is twice as high and that of primary phenols twice as low, when compared to those produced by cells treated with 80 microM BP. The AHH activity drastically modifies the overall rate of the BP metabolism but does not affect the qualitative pattern of the excreted metabolites. The overall metabolism of [3H]BP by the cell culture can easily be estimated by measuring the release of the tritiated water from the substrate into the culture medium.

Organ Specificity of Induction of Activating and Inactivating Enzymes by Cigarette Smoke and Cigarette Smoke Condensate

Inhalation of cigarette smoke specifically induces the rat lung and kidney aryl hydrocarbon hydroxylase (AHH) in less than 4 h. The epoxide hydratase (EH) and the glutathione S-transferase are not significantly modified by a similar treatment in any of the rat tissues. Compared to the kidney AHH, the lung hydroxylase is 3--4 times more sensitive to small concentrations of cigarette smoke and seems to have a longer biological half-life. In both tissues, the induced AHH presents the same in vitro sensitivity to various inhibitors as a polycyclic hydrocarbon induced AHH. In primary fetal rat liver cell culture, the cigarette smoke condensate fractions (CSCF) induce both the AHH and EH activity. Nevertheless, the AHH activity responds faster and to lower concentrations of CSCF than the EH activity. The liver cell culture constitutes a unique tool for a comparative study of the AHH and EH induction mechanism. Low concentration (10 muM) of benz(a)anthracene induces only the AHH activity while trans-stilbene oxide enhances selectively the EH activity. Appropriate concentrations of CSCF or of phenobarbital (PB) determine a parallel induction of both enzymes. The results are discussed on the basis of (a) the existence of specific mechanisms of AHH regulation in the lung and in the kidney and (b) the existence of coordinated or independent biochemical control of the AHH and EH activity.

Comparison of aryl hydrocarbon hydroxylase and epoxide hydratase. Induction in primary fetal rat liver cell culture

The activity of aryl hydrocarbon hydroxylase (AHH) and/or epoxide hydratase (EH) is induced in primary fetal rat liver cell culture by benz-[alpha]anthracene (BA), phenobarbital (PB), cigarette smoke condensate (CSC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and trans-stilbene oxide (TSO). The response of the two enzymes to the different chemicals varies as follows: (a) AHH is induced by lower concentrations of BA, PB and CSC than those required to significantly induce EH; (b) AHH is selectively induced by TCDD and by low BA concentrations; (c) the kinetics of AHH induction by BA, PB and CSC is faster than that of EH; (d) TSO is a selective inducer of EH. As described earlier for AHH, RNA and protein synthesis and the continuous presence of the inducer are required in the early phases of EH induction. Later when the EH activity has reached a plateau, intact RNA and protein synthesis is not necessary to maintain the enzyme at its optimal value. The removal of the inducer determines a decay of the EH activity, allowing the estimation of a biological tau 1/2 of about 72 h. TSO prevents the AHH induction by PB, but not that mediated by BA and CSC. Added together with PB, BA, CSC or PB plus BA, TSO induces the EH activity in a more than additive manner. This effect is only seen after 6 days of continuous treatment. These results indicate that in this tissue culture model, the mechanism of AHH and EH induction can clearly be dissociated.

Induction of liver microsomal cytochrome P-450 isozymes by 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide

1. 1-(2-Chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (RP 52028), an antagonist of peripheral type benzodiazepine binding sites, a potential anticonvulsant, has been shown to have an inducing effect on drug-metabolizing enzymes. 2. RP 52028 was administered orally at 20-800 mg/kg for 1 week, and enzymic activities were determined using a panel of substrates. Western blot analyses were performed using several specific polyclonal and monoclonal antibodies directed against cytochrome P-450 isozymes. 3. RP 52028 appears to be an inducer of cytochrome P-450 II B1 (P-450b) and related enzymic activities; i.e. benzphetamine, ethylmorphine and aminopyrine demethylation.

Differences in the biochemical properties of aldrin epoxidase, a cytochrome P-450-dependent monooxygenase, in various tissues

Aldrin epoxidase, a cytochrome P-450-dependent monooxygenase, was studied in the lung and kidney of male rats. The sensitivity of the liver enzyme activity to different chemicals in vitro was influenced by the treatment of the animals with phenobarbital or methylcholanthrene. These results confirm that more than one form of cytochrome P-450 supports aldrin epoxidase in the liver. The lung and kidney aldrin epoxidase activity was not modified by the administration of chemical inducers to the rats. In vitro, the lung and kidney aldrin epoxidase activities were activated by tetrahydrofurane and progesterone, respectively. The results obtained from the lung and kidney indicate that one single species of cytochrome P-450, associated with aldrin epoxidase, exists in these organs, but it may be a different type, or regulated in a different manner in these tissues.

MOLECULAR REGULATORY MECHANISMS OF NYCTOHEMERAL RHYTHMS IN HEPATIC CELL DIVISION AND FUNCTION

ABSTRACT Under various physiological conditions, cell division function (tested by nuclear-RNA synthesis, DNA synthesis, labelling index after tritiated thymidine administration, mitotic index) and specific hepatic function (mainly assayed by cholesterol-7α-hydroxylase activity) have been compared in the liver of the rats. In adult and young rat livers, with or without partial hepatectomy, the triggering of these two different functions presents a mutually exclusive relationship and a very pronounced circadian rhythm : the derepression of the genes coding enzymes of these two functions never occurs simultaneously and thus takes place at two different moments of the nycthemeron. The kinetics of these two function evolutions could be related by the chalone regulatory mechanisms where corticoids act as effectors.