Expression Of Cytochrome P450 mRNAs Research Articles

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Attenuation of carbon tetrachloride toxicity in acute hepatosteatosis induced by intake of a high carbohydrate diet after food deprivation (959.1)

Hepatic fat accumulation constitutes a major component in the pathogenesis of liver injury. We determined the impact of lipid deposition in liver on cytochrome P‐450 (CYP) system and CCl4‐induced hepatotoxicity. Rats were fasted for 48 h, and then provided with a high‐carbohydrate/fat‐free diet (FH) or a normal diet (FN) for 48 h. Hepatic triglyceride level was not different between the normal control and the FN group, but 4 times greater in the FH group, which was consistent with the histopathological observation. FN intake resulted in a small increase in microsomal p‐nitrophenol hydroxylase, p‐nitroanisole O‐demethylase, and erythromycin N‐demethylase activities, but aminopyrine N‐demethylase was decreased. The microsomal enzyme activities determined were all inhibited markedly by FH intake. Immunoblotting analysis showed that proteins of CYP2E1, 1A, 3A, and 2B1/2 were decreased in the rats fed FH. Expression of CYP mRNAs was also down‐regulated. A dose of CCl4 was administered to the rats fed the different diets. The resulting liver injury was significantly lower in the FH group than in the FN group as determined by elevation of serum enzyme activities and histopathological examination. The results indicate that acute hepatosteatosis can precipitate erratic responses to various endogenous and exogenous substances via alterations in hepatic CYP‐mediated metabolizing capacity.

Regulation of MicroRNA Expression by Rifampin in Human Hepatocytes

Rifampin causes drug interactions by altering hepatic drug metabolism. Because microRNAs (miRNAs) have been shown to regulate genes involved in drug metabolism, we determined the effect of rifampin on the expression of hepatic miRNAs. Primary human hepatocytes from seven subjects were treated with rifampin, and the expression of miRNA and cytochrome P450 (P450) mRNAs was measured by TaqMan assays and RNA-seq, respectively. Rifampin induced the expression of 10 clinically important and 13 additional P450 genes and repressed the expression of 9 other P450 genes (P < 0.05). Rifampin induced the expression of 33 miRNAs and repressed the expression of 35 miRNAs (P < 0.05). Several of these changes were highly negatively correlated with the rifampin-induced changes in the expression of their predicted target P450 mRNAs, supporting the possibility of miRNA-induced regulation of P450 mRNA expression. In addition, several other miRNA changes were positively correlated with the changes in P450 mRNA expression, suggesting similar regulatory mechanisms. Despite the interindividual variability in the rifampin effects on miRNA expression, principal components analysis clearly separated the rifampin-treated samples from the controls. In conclusion, rifampin treatment alters miRNA expression patterns in human hepatocytes, and some of the changes were correlated with the rifampin-induced changes in expression of the P450 mRNAs they are predicted to target.

Modified expression of cytochrome P450 mRNAs by growth hormone in mouse liver

The expression of eight mouse hepatic cytochrome P450s (P450s) genes was investigated at the mRNA level in relation with the pattern of growth hormone (GH) administration. The constitutive expression of five sex-dependent P450s was sexually dimorphic, namely female > male for CYP2A4, CYP2B9, CYP2B10, and CYP3A41, and male > female for CYP2D9. In mice neonatally treated with monosodium l-glutamate to produce GH-deficiency, GH was found to be an essential factor with GH archetype as a determinant in the regulatory mechanism of hepatic CYP2D9 and CYP3A41 expression, and GH was shown to be a repressive factor for the constitutive expression in females. Implantation with micro-osmotic pump containing GH (to yield a constant release of GH to mimic the plasma GH profile in females) to male mice increased CYP2A4, CYP2B9, CYP2B10, and CYP3A41, but decreased CYP2D9, expression to female levels, while conversely, twice-daily administration of GH (to produce the so-called male pattern of plasma GH levels) to female mice resulted in the repression of female-specific, CYP2B9 and CYP3A41, as well as female-predominant, CYP2A4 and CYP2B10, expression, and induction of male-specific CYP2D9 expression. Thus, the sex-dependent plasma GH profile (referred to hereafter as the GH archetype) was a decisive factor for the expression of sex-specific P450 genes in adult mouse liver. On the other hand, the regulation of CYP1A2, CYP2C29, and CYP3A11 expression was either sex-independent or GH archetype-independent, considering the comparable levels between sexes of the constitutive expression and GH-inducible expression of these isoforms. Moreover, the observations suggested for the first time that the expression of CYP2B9 and CYP2A4 was not entirely GH-independent, but rather involved an imprinting GH-related factor that participated in the regulatory mechanism of P450 expression in females.

Hepatic Cytochrome P450 Gene Regulation during Endotoxin-Induced Inflammation in Nuclear Receptor Knockout Mice

Inflammatory agents such as lipopolysaccharide (LPS) down-regulate the hepatic expression of many cytochrome P450 (P450) mRNAs and proteins. Previous studies suggested that suppression of some P450 mRNAs could involve the regulation or modulation of the nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) or pregnane X receptor (PXR). To determine the involvement of these receptors in P450 down-regulation, PPARalpha knockout (KO), PXR KO, and appropriate wild-type (WT) mice were administered either saline or 1 mg/kg LPS. Hepatic mRNA and protein expression of several P450 isoforms, interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF) alpha, alpha1-acid glycoprotein (AGP), and fibrinogen (FBG) were examined 16 h later. LPS administration significantly decreased the hepatic expression of CYP1A2, 2A5, 2C29, 2E1, 3A11, 4A10, and 4A14 mRNAs in both groups of PPARalpha and PXR mice, whereas CYP3A13 mRNA was increased slightly in PPARalpha WT and KO mice, but not in PXR mice. Effects of LPS administration on mouse hepatic P450 proteins (probed using rat P450 2C, 3A, 4A, and 2E antibodies) were consistent with mRNA results in most cases. LPS treatment significantly increased IL-1beta, IL-6, TNFalpha, AGP, and FBG mRNA in both PPARalpha and PXR mice, with the greatest effect observed with TNFalpha. Because decreases in P450 mRNA expression were essentially identical in both WT and KO mice for both nuclear receptors, these data indicate that down-regulation of P450 during inflammation does not require the nuclear receptors PPARalpha and PXR.

Mode of Action of Chlorinated Ethylenes on the Expression of Rat Cytochrome P450 forms and Specificity in the Metabolic Activation of CEs by CYPs.

Chlorinated ethylenes (CEs) including tetrachloroethylene (PCE), trichloroethylene (TCE) and 1,1-dichloroethylene (DCE) were comparatively evaluated for their effects on the expression of cytochrome P450 (CYP) forms of subfamilies 1A, 2B, 2E and 3A as well as their relative suitability as substrates of these CYPs. The magnitudes of inhibition of the enzyme activities were as follows in descending order: 1,1-DCE > TCE > PCE against hepatic CYPs and PCE > 1,1-DCE > TCE against pulmonary CYP2B1. These organ-specific profiles in the sensitivities to the adverse effects of CEs were partly attributable to the differential expression patterns of CYP forms by which they were metabolically activated. The expression of hepatic and pulmonary CYP2B mRNA was severely suppressed in the presence of 1,1-DCE during the entire observation period until 30 hr after the CE-treatment, in marked contrast to the temporarily enhanced expression at 6 hr followed by a moderate suppression in the cases of PCE and TCE with the trough values being observed at 18 hr. In addition to CYP2B, 1,1-DCE in advance of the transcriptional stage, when simultaneously treated with phenobarbital, also exclusively suppressed CYP2E1. These general suppressive effects of 1,1-DCE on the expression of divergent CYP mRNAs in vivo resembled the published findings in primary cultured hepatocytes treated with inflammatory cytokines such as IL-1β, TNF-α and IL-6, implying the highly inflammatory nature of 1,1-DCE.

Comparative Study on Correlation between Chemical Structure and Effect on Expression of Cytochrome P450 mRNAs in Rat among Chlorinated Ethylenes, Tetrachloroethylene, Trichloroethylene, 1, 1-Dichloroethylene.

Three polychlorinated ethylenes (CEs), tetrachloroethylene (PCE), trichloroethylene (TCE) and 1, 1-dichloroethylene (DCE), were comparatively studied for their effects on the expression of cytochrome P450 (CYP) mRNA forms in the liver and lung using 7-weeks-old male Wistar rats. The animals were i.p. inoculated with 0.5 g/kg of the individual CEs and sacrificed at 6-hr intervals for 30 hr for the determination of the mRNA levels of hepatic and pulmonary CYP2B and hepatic CYP2E1. A 3.5-fold increase in the expression of hepatic CYP2B mRNA was noted transiently at 6 hr in the presence of PCE. In contrast, 1, 1-DCE was suppressive and started within 6 hr and lasted for more than 30 hr, with a trough value of 15% of the control being observed around 12 to 18 hr, while there was no marked effect observed in the case of TCE-treatment. The expression of pulmonary CYP2B mRNA was severely suppressed in the presence of 1, 1-DCE during the entire observation period as was the case with its hepatic counterpart, while the temporarily enhanced expression of mRNA at 6 hr by PCE and TCE was followed by a moderate suppression, with the trough values of ca. 80 and 65% of the control, respectively, at 18 hr. Concerning the hepatic CYP2E1, the expression of mRNA was adversely affected by all the CEs with a descending order of magnitude of 1, 1-DCE, TCE and PCE with peak inhibition values of 85%, 50% and 35%, respectively. The nonselective suppressive effect of 1, 1-DCE on the expression of divergent CYP mRNAs was well correlated with the IL-1β-dependent suppression of various types of CYP mRNA in primary cultured hepatocytes previously reported. Although PCE and TCE are under stringent legislative restriction in Japan as environmental pollutants owing to their relatively stable natures, they are less toxic in terms of the inhibition of the mRNA expression of CYP forms in acute phases than short-lived 1, 1-DCE, which is inflammatory to the host animals; the shorter the environmental life span (1, 1-DCE > TCE > PCE), the more severe the acute toxicity.

Induction of cytochrome P450 CYP6B7 and cytochrome b5 mRNAs from Helicoverpa armigera (Hubner) by pyrethroid insecticides in organ culture.

An organ culture system derived from Helicoverpa armigera has been used to study the expression of cytochrome P450 and cytochrome b5 mRNAs. Northern analysis showed that levels of the mRNAs for cytochrome P450s, CYP6B2, CYP6B6 and CYP6B7, and cytochrome b5 in control tissue were commensurate with those in the tissue of whole larvae. Substantial induction of cytochrome P450, CYP6B7 and cytochrome b5 mRNAs by alpha-pinene, and the pyrethroids, fenvalerate, cypermethrin and permethrin were observed in fat body culture. Neither mRNA was induced, either in midgut or integument organ cultures. In contrast, the relatively water-soluble compound phenobarbital, could induce CYP6B7 mRNA but not cytochrome b5 mRNA in fat body cultures. As for pyrethroids, phenobarbital had no effect on the other tissues in culture. These results confirm a previous conclusion that pyrethroids could induce CYP6B7 mRNA, which was based upon a very slight induction observed in living insects. Because many cytochrome P450 substrates can act as their inducers, these results support a previous conclusion that CYP6B7 could be the enzyme that is involved in pyrethroid resistance in H. armigera.

Characterization of cytochrome P450 and glutathione S-transferase activity and expression in male and female ob/ob mice.

To characterize the effect(s) of gender, age (glycemic status) and obese state, on hepatic biotransformation activities, expression of cytochrome P450 (CYP450) mRNAs and glutathione transferase activity in the ob/ob mouse. Male and female, ob/ob or ob/+ mice were killed at 3-4 months or 7-8 months of age. Hepatic microsomes, cytosol and RNA were prepared from each animal. Male and female ob/ob and ob/+ mice, 3-4 or 7-8 months of age. CYP450 form-specific activities of CYP1A1/1A2, CYP3A and CYP2B were estimated by determining the 0-dealkylation of alkoxyresorufin substrates (ethoxy-EROD, benzoxy-BROD and pentoxy-resorufin, PROD, respectively). CYP2E1-dependent, 4-nitrophenol hydroxylase (PNP-OH) and CYP3A-dependent erythromycin N-demethylase (ERY-DM) were also measured in hepatic microsomes. CYP1A2, CYP2E1 and CYP3A protein in microsomal fractions was determined by ELISA. Glutathione transferase activity (GST) was determined in hepatic cytosol and CYP1A2 and CYP2E1 mRNA was estimated by Northern blot analysis. Female mice, regardless of glycemic status, showed an obesity enhanced level of CYP2E1-dependent PNP-OH activity and CYP2E1 protein as shown by ELISA. These increases were observed to be independent of the diabetic state, since 7-8 month-old mice had blood glucose levels identical to lean mice. The mRNA level of CYP2E1 in female mice also exhibited age-and obesity-influenced decreases in expression. No significant differences in CYP2E1 activity or expression were observed in male mice. CYP3A-dependent ERY-DM activity was significantly higher in young males, regardless of phenotype. CYP3A and CYP2B activities did not differ among any animals; however, CYP1A activity, while depressed in obese animals of both genders, was significantly different in old animals. Glutathione S-transferase activity was lower in obese male mice, whereas no difference was observed between lean and obese females This study supports earlier observations in man and rats that the obese state produces alterations in the expression of important oxidation and conjugation pathways. In addition, this report more thoroughly examines the role of gender and glycemic status on biotransformation activities in the ob/ob mouse as demonstrated by increased CYP2E1 protein and CYP2E1-dependent activity in obese females, decreased CYP1A2 protein and CYP1A2-dependent activity in obese animals, and obesity had no effect of glutathione transferase in female mice, in contrast with the previously reported obesity-dependent decrease of this activity in male mice.

Expression of Cytochrome P450 mRNAs in the Colon and the Rectum in Normal Human Subjects

The expression of mRNA for three isozymes of cytochrome P450, CYP1A1, 1A2 and 3A3, was examined in biopsied samples of colon and rectum from five healthy subjects. Using RT-PCR, it was possible to detect the three CYP mRNA species at both sites in all samples examined. The expression of each CYP mRNA was site-specific in that rectum had higher levels of CYP1A1 mRNA than colon, while colon had higher levels of CYP3A3 mRNA than in rectum. In contrast, there was no consistent trend in CYP1A2 mRNA expression between the two sites. Variability of alimentary CYP expression may contribute to individual differences in susceptibility to carcinogens and/or drugs.

Decreased Expression of Cytochrome P450 mRNAs and Related Steroid Hydroxylation Activities in Hepatic Hyperplastic Nodules in Male F344 Rats

To explore the mechanism(s) underlying the relationship between expression of cytochrome P450 (CYP) enzymes and growth of hepatic hyperplastic nodules, mRNA of CYP1A1, 1A2, 2B1, 2B2, 2C6, 2C11, and 3A1, and CYP enzyme-mediated hydroxylation of testosterone (T), I 7β-estradiol (E), and corticosterone (C) were determined in a group of selected large nodules from livers of male F344 rats given the modified Solt-Farber resistance protocol and compared with control and surrounding tissue. Slot-blot analysis with specific oligonucleotides showed little or no mRNA of CYP2B1, 2B2, and 2C6 in nodules, surrounding tissue, and control liver without PB treatment. Constitutive expression, as many endogenous CYP1A2, 2C11, and 3A1 in nodules was from 2 to 10 times less in nodules than in control liver. PB treatment increased mRNA of CYP21, 2B2, 2C6, 2X11, and 3A1 in control and surrounding tissue substantially, but was 3 to 11 times less in the nodules than in the control liver. Messenger RNA of CYP1A1 was not detected in either nodules or control livers independent of PB treatment. HPLC determination demonstrated a general pattern of a decrease on CYP enzyme-mediated hydroxylation of T, E, and C in the nodules relative to the control liver. The differences between nodules and control liver were usually 3 to 10-fold for the 12 metabolites detected although smaller decreases were observed for a few metabolites in some nodules. PB increased the rate of hydroxylation for 11 of the 12 metabolites and resulted in 2 additional metabolites of both T and E in control liver and some nodules. However, the general pattern of lower hydroxylation activities in the nodules relative to the surrounding tissue remained the same as that in the non-PB group. These observations are consistent with the hypothesis that intracellular kinetics of endogenous CYP enzyme substrates may differ in some nodules, relative to the surrounding tissue, as a result of decreased expression of CYP genes. This difference in CYP expression pattern in turn may contribute to the selective growth and progression to cancer of certain nodules, as many endogenous CYP enzyme substrates are modulators of DNA and RNA synthesis and cellular growth and differentiation.

Sex-specific, growth hormone-regulated transcription of the cytochrome P450 2C11 and 2C12 genes.

Growth hormone (GH) differentially regulates the expression of several male-specific and female-specific liver cytochrome P450 mRNAs as a function of its sex-dependent ultradian secretory pattern. Pulsatile GH release stimulates expression of the male-specific P450 2C11, while a continuous GH secretion pattern suppresses expression of 2C11 and stimulates the expression of the female-specific P450 2C12. To help define the level at which GH regulates the expression of 2C11 and 2C12 mRNA, liver nuclear RNA samples isolated from rats differing in GH status were analyzed for 2C11 and 2C12 hnRNAs by hybridization to 2C11 and 2C12 gene-specific exonic oligonucleotide probes, as well as exon/intron junction probes. The 2C11 and 2C12 hnRNAs were found to be responsive to circulating GH profiles in a manner indistinguishable from the corresponding mature, cytoplasmic mRNAs, with no 2C12 mRNA precursors found in untreated male or hypophysectomized female liver nuclei, and no 2C11 mRNA precursors in untreated female or hypophysectomized male liver nuclei. Thus, transport of 2C11 and 2C12 RNA to the cytoplasm and cytoplasmic mRNA stability are unlikely to be important GH-regulated control points for sex-specific P450 RNA expression. Run-on transcription analysis further established that GH regulates the sex-specific expression of the 2C11 and 2C12 genes at the level of transcript initiation. Transcription was also shown to be the major step for regulation of the male-specific P450 2A2 RNA, whose expression, unlike 2C11, is not obligatorily dependent on pulsatile GH release. In vitro footprinting analysis of 2C11 and 2C12 promoter fragments incubated with liver nuclear proteins isolated from rats differing in GH status revealed several sex- and GH-dependent differences in DNase cleavage patterns ("hypersensitivity sites"), demonstrating that GH can regulate specific protein-DNA interactions in the 5'-flanking sequences of these two genes. In vitro transcription assays driven by 2C11 and 2C12 5'-flanking DNA sequences fused to TATAA box-G-less cassette template constructs did not, however, faithfully mimic the sex-specific transcription of the 2C11 and 2C12 genes, indicating that additional cis-elements or trans-acting factors may be required to achieve the transcriptional regulation of these genes that occurs in vivo.

Expression of cytochrome P-450 mRNAs in steroidogenesis of adrenocortical adenomas from patients with primary aldosteronism

We studied the contents of aldosterone and cortisol (F) and the expression of mRNA of cytochrome P-450 for side-chain cleavage ( P-450 scc), 17α-hydroxylase ( P-450 c17), 21-hydroxylase ( P-450 c21) and 11β-hydroxylase ( P-450 c11) in adrenocortical adenomas from three patients with primary aldosteronism. The aldosterone content was significantly higher in adrenocortical adenomas than in normal adrenal glands, while F content in adenomas was similar to the level in normal adrenal glands. The aldosterone-producing adenomas showed a markedly higher level of P-450 c11 mRNA, a slightly but not significantly increased level of P-450 c21 mRNA and a significantly decreased level of P-450 c17 mRNA, compard with those in normal adrenal glands. The expression of P-450 scc mRNA in adenomas was similar to the level in normal adrenal glands. These results suggested that the renin-independent overproduction of aldosterone in adrenocortical adenomas from the patients with primary aldosteronism results from increasing expression of the mRNA for P-450 c11 and decreasing expression of the mRNA for P-450c17.