Immunolocalisation of cytochrome P-450 3A enzymes in human breast carcinoma: relationship with tumour differentiation and steroid receptors.

Cytochrome P-450 3A enzymes belong to the most abundant subfamily of the cytochrome P-450 system. They are predominantly found in the liver where they metabolize numerous drugs and endogenous substances such as oestrogens. However, they are also expressed by normal and tumoural extrahepatic tissues. Accordingly, immunolocalization was assessed in malignant breast tumours (n=32) and normal counterparts, by using a monoclonal antibody that recognizes all human CYP3A proteins. We investigated a potential relation between expression of CYP3A protein expression, the degree of tumour differentiation assessed by the histological grade and the proliferation index assessed by Ki-67 immunostaining. Immunodetection of CYP3A was observed in 27 of the 32 tumours analyzed (84%). A focal staining was also observed in the adjacent normal breast tissue in 33% of the samples, but expression was always fainter than in tumours. A significant negative association was found between CYP3A and the proliferation index, but there was no relation with receptor status or tumour differentiation. While CYP3A protein expression can be found in normal breast tissues, these data highlight higher and more frequent CYP3A in malignant breast cells. Such expression in malignant breast cells appears inversely related to the proliferation index whereas no relation is found with tumour differentiation.

Early down-regulation of cytochrome P450 3A and 2E1 in the regenerating rat liver is not related to the loss of liver mass or the process of cellular proliferation.

AIMS/BACKGROUND: Conflicting data have been reported concerning the modification of cytochrome P450 expression in the regenerating liver. Ligation of branches of the portal vein (PBL) perfusing 70% of the liver parenchyma, which produces regeneration and atrophy within the same liver, constitutes an ideal model to study the relative specificity of the early events in the regenerating liver and their relationship to the loss of liver mass. METHODS: In this PBL model and in sham models, we studied the expression and the metabolic activities of two major cytochromes, CYP3A and CYP2E1, and the expression of inducible nitric oxide synthase protein (iNOS). They were simultaneously measured in the atrophying and regenerating liver lobes following PBL using Western Blot and HPLC methods. RESULTS: The metabolic activities of both cytochromes were transiently and simultaneously down-regulated in the regenerating and atrophying lobes during the first 2-5 h after PBL. No significant modification was observed at the protein level. In contrast, iNOS protein was significantly induced in both lobes. Similar results were observed after sham operation. CONCLUSIONS: The reduction of these CYP activities in both lobes after PBL and in sham livers suggests that other mechanisms than the regenerating process itself or the reduction of the liver mass might account for such down-regulation during the early phase of liver regeneration. The activation of nitric oxide (NO) and/or pro-inflammatory cytokine production provides clues to pathways liable to affect the CYP activities in the regenerating liver.

CYP 3A proteins are expressed in human neutrophils and lymphocytes but are not induced by rifampicin.

Cytochrome P-450 3A (CYP 3A) enzymes, the prominent subfamily in the cytochrome system, are expressed in various extrahepatic tissues. Until now, their expression has been demonstrated in human polymorphic neutrophils (PMNs) but not in lymphocytes using immunohistochemistry and immunoblot analysis. Moreover, their potential modulation has not been determined yet. To study such an expression in different peripheral blood cell populations, rifampicin (600 mg/day during 6 days) was given to 8 healthy subjects. PMNs and lymphocytes were isolated by centrifugation of whole white blood cell fractions using Ficoll gradients before drug administration, immediately after, and 3 days after drug withdrawal. PMN and lymphocyte smears and homogenates were subjected to immunostaining and immunoblotting, respectively, with a mouse monoclonal antibody recognizing all CYP 3A proteins. These proteins were quantified by densitometric analysis. Before and after rifampicin administration, a positive cytoplasmic staining was observed in all PMNs and in about 50% of lymphocytes. CYP 3A expression in lymphocytes was further confirmed by positive immunoblots for lymphocyte homogenates. Neither in PMNs nor in lymphocytes, induction of CYP 3A protein expression was observed after rifampicin treatment despite overall induction of CYP 3A activity assessed by the urinary excretion of 6beta-hydroxycortisol. These results demonstrate that CYP 3A proteins are constitutively expressed not only in PMNs but also in lymphocytes. However, in both cell lineages CYP 3A protein expression was not induced by rifampicin.

14C-Propoxyphene demethylation in the rat. An example of differences between liver and intestinal drug-presystemic metabolism.

Presystemic metabolism is believed to occur mainly in the liver with some minor intestinal participation. The aim of this study was to investigate the respective part of each of these two organs in the metabolism of the analgesic d-propoxyphene (DP). Pharmacological doses of DP were given in the duodenum (ID), the portal vein (IP), and the femoral vein (IV) of male Wistar rats. A tracer dose of 14C-DP was also administered either in IV, IP, or ID as well as in hepatectomized rats or rats with bile duct diversion. In vitro demethylation occurring in liver and intestinal microsomes was also studied. Absolute DP bioavailability obtained after oral administration was two times higher than that observed after portal administration (48.9% vs. 23.2%, respectively), an result opposite (i.e. a lower bioavailability) of that expected on the basis of the existence of a liver enzyme saturation phenomenon. The 14CO2 cumulative excretion after 14C-DP administration was significantly lower after IV or ID administration than after injection in the portal vein as a bolus or within 20 min. The biliary excretion of the labeled compound varied in the opposite direction, being greater after IV or ID than after IP administration, suggesting that the metabolism of DP in the liver is influenced by an extrahepatic transformation. This most likely occurs in the gut since the production of 14CO2 after IV administration was similar to that after ID administration. This transformation did not prohibit DP detection in the systemic blood but was sufficient to increase the part eliminated with bile and to decrease the part demethylated into NP. Demethylation mainly occurs in the liver since the production of 14CO2 was nearly abolished in hepatectomized rats. Furthermore, microsomes of hepatic but not of intestinal origin were able to demethylate DP. Our data suggest that the transformation of DP occurring in gut after oral administration is responsible for changes in the hepatic metabolism of the drug.

Phenytoin hydroxylation in a healthy Caucasian population: bimodal distribution of hydroxyphenytoin urinary excretion.

The phenytoin hydroxylation index was studied in 122 unrelated Caucasian volunteers. Following a 100 mg oral dose of phenytoin, phenytoin and hydroxyphenytoin were measured in urine from 0-32 hr after administration. As phenytoin was not found in all urine collections, a phenytoin hydroxylation index was expressed as follows: Phenytoin hydroxylation index = amount of phenytoin administered/0-32 hr urinary output of hydroxyphenytoin. Phenytoin hydroxylation index values appear to be bimodally distributed, 92% of the population showing a mean (+/- S.E.M.) value of 0.639 +/- 0.099 and 8% a mean (+/- S.E.M.) value of 1.001 +/- 0.180 (log10 values). These results are in favour of the existence of a phenytoin genetic polymorphism. Since misuse of urinary metabolite excretion data in drug metabolism studies is a well-known phenomenon, our data emphasize the need for future population studies on phenytoin pharmacokinetics as well as on CYP2C9 genotyping before concluding about existence of a phenytoin genetic polymorphism.

Effects of simvastatin and pravastatin on 6 beta-hydroxycortisol excretion, a potential marker of cytochrome P-450 3A.

The influence of two inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, simvastatin and pravastatin, on the level of urinary 6 beta-hydroxycortisol/17-hydroxycorticosteroids (6 beta-OHC/17-OHCS) ratio was determined in two groups of normolipidemic Caucasian subjects (n = 18 and n = 14, respectively). The 6 beta-OHC/17-OHCS ratio increased significantly after simvastatin administration (20 mg day-1 for 17 days) (P = 0.0125) whereas no modification was observed after pravastatin administration (20 mg day-1 during 17 days). As the level of 6 beta-OHC/17-OHCS ratio is a function of cytochrome P-450 3A activity, these results suggest that in Caucasian subjects, simvastatin but not pravastatin could be a weak inducer of cytochrome P-450 3A. This contrasting effect could be related to the major pharmacological differences existing between these two drugs.