Increase In CYP1A2 Research Articles

Assessment of Herb-Drug Interaction Potential of Five Common Species of Licorice and Their Phytochemical Constituents

The dried roots and rhizomes of Glycyrrhiza species (G. glabra, G. uralensis and G. inflata), commonly known as licorice, have long been used in traditional medicine. In addition, two other species, G. echinata and G. lepidota are also considered “licorice” in select markets. Currently, licorice is an integral part of several botanical drugs and dietary supplements. To probe the botanicals’ safety, herb-drug interaction potential of the hydroethanolic extracts of five Glycyrrhiza species and their key constituents was investigated by determining their effects on pregnane X receptor, aryl hydrocarbon receptor, two major cytochrome P450 isoforms (CYP3A4 and CYP1A2), and the metabolic clearance of antiviral drugs. All extracts enhanced transcriptional activity of PXR and AhR (>2-fold) and increased the enzyme activity of CYP3A4 and CYP1A2. The highest increase in CYP3A4 was seen with G. echinata (4-fold), and the highest increase in CYP1A2 was seen with G. uralensis (18-fold) and G. inflata (16-fold). Among the constituents, glabridin, licoisoflavone A, glyasperin C, and glycycoumarin activated PXR and AhR, glabridin being the most effective (6- and 27-fold increase, respectively). Licoisoflavone A, glyasperin C, and glycycoumarin increased CYP3A4 activity while glabridin, glyasperin C, glycycoumarin, and formononetin increased CYP1A2 activity (>2-fold). The metabolism of antiretroviral drugs (rilpivirine and dolutegravir) was increased by G. uralensis (2.0 and 2.5-fold) and its marker compound glycycoumarin (2.3 and 1.6-fold). The metabolism of dolutegravir was also increased by G. glabra (2.8-fold) but not by its marker compound, glabridin. These results suggest that licorice and its phytochemicals could affect the metabolism and clearance of certain drugs that are substrates of CYP3A4 and CYP1A2. Supplemental data for this article is available online at https://doi.org/10.1080/19390211.2022.2050875 .

Zuotai (β-HgS)-containing 70 Wei Zhen-Zhu-Wan differs from mercury chloride and methylmercury on hepatic cytochrome P450 in mice.

Background: Zuotai (mainly β-HgS)-containing 70 Wei-Zhen-Zhu-Wan (70W, Rannasangpei) is a famous Tibetan medicine for treating cardiovascular and gastrointestinal diseases. We have shown that 70W protected against CCl 4 hepatotoxicity. CCl 4 is metabolized via cytochrome P450 (CYP) to produce reactive metabolites. Whether 70W has any effect on CYPs is unknown and such effects should be compared with mercury compounds for safety evaluation. Methods: Mice were given clinical doses of 70W (0.15-1.5 g/kg, po), Zuotai (30 mg/kg, po), and compared to HgCl 2 (33.6 mg/kg, po) and MeHg (3.1 mg/kg, po) for seven days. Liver RNA and protein were isolated for qPCR and Western-blot analysis. Results: 70W and Zuotai had no effects on hepatic mRNA expression of Cyp1a2, Cyp2b10, Cyp3a11, Cyp4a10 and Cyp7a1, and corresponding nuclear receptors [aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-α (PPARα); farnesoid X receptor (FXR)]. In comparison, HgCl 2 and MeHg increased mRNA expression of Cyp1a2, Cyp2b10, Cyp4a10 and Cyp7a1 except for Cyp3a11, and corresponding nuclear receptors except for PXR. Western-blot confirmed mRNA results, showing increases in CYP1A2, CYP2B1, CYP2E1, CYP4A and CYP7A1 by HgCl 2 and MeHg only, and all treatments had no effects on CYP3A. Conclusions: Zuotai and Zuotai-containing 70W at clinical doses had minimal influence on hepatic CYPs and corresponding nuclear receptors, while HgCl 2 and MeHg produced significant effects. Thus, the use of total Hg content to evaluate the safety of HgS-containing 70W is inappropriate.

Zuotai (β-HgS)-containing 70 Wei Zhen-Zhu-Wan differs from mercury chloride and methylmercury on hepatic cytochrome P450 in mice

Background: Zuotai (mainly β-HgS)-containing 70 Wei-Zhen-Zhu-Wan (70W, Rannasangpei) is a famous Tibetan medicine for treating cardiovascular and gastrointestinal diseases. We have shown that 70W protected against CCl 4 hepatotoxicity. CCl 4 is metabolized via cytochrome P450 (CYP) to produce reactive metabolites. Whether 70W has any effect on CYPs is unknown and such effects should be compared with mercury compounds for safety evaluation. Methods: Mice were given clinical doses of 70W (0.15-1.5 g/kg, po), Zuotai (30 mg/kg, po), and compared to HgCl 2 (33.6 mg/kg, po) and MeHg (3.1 mg/kg, po) for seven days. Liver RNA and protein were isolated for qPCR and Western-blot analysis. Results: 70W and Zuotai had no effects on hepatic mRNA expression of Cyp1a2, Cyp2b10, Cyp3a11, Cyp4a10 and Cyp7a1, and corresponding nuclear receptors [aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-α (PPARα); farnesoid X receptor (FXR)]. In comparison, HgCl 2 and MeHg increased mRNA expression of Cyp1a2, Cyp2b10, Cyp4a10 and Cyp7a1 except for Cyp3a11, and corresponding nuclear receptors except for PXR. Western-blot confirmed mRNA results, showing increases in CYP1A2, CYP2B1, CYP2E1, CYP4A and CYP7A1 by HgCl 2 and MeHg only, and all treatments had no effects on CYP3A. Conclusions: Zuotai and Zuotai-containing 70W at clinical doses had minimal influence on hepatic CYPs and corresponding nuclear receptors, while HgCl 2 and MeHg produced significant effects. Thus, the use of total Hg content to evaluate the safety of HgS-containing 70W is inappropriate.

Zuotai (β-HgS)-containing 70 Wei Zhen-Zhu-Wan differs from mercury chloride and methylmercury on hepatic cytochrome P450 in mice.

Background: Zuotai (mainly β-HgS)-containing 70 Wei-Zhen-Zhu-Wan (70W, Rannasangpei) is a famous Tibetan medicine for treating cardiovascular and gastrointestinal diseases. We have shown that 70W protected against CCl 4 hepatotoxicity. CCl 4 is metabolized via cytochrome P450 (CYP) to produce reactive metabolites. Whether 70W has any effect on CYPs is unknown and such effects should be compared with mercury compounds for safety evaluation. Methods: Mice were given clinical doses of 70W (0.15-1.5 g/kg, po), Zuotai (30 mg/kg, po), and compared to HgCl 2 (33.6 mg/kg, po) and MeHg (3.1 mg/kg, po) for seven days. Liver RNA and protein were isolated for qPCR and Western-blot analysis. Results: 70W and Zuotai had no effects on hepatic mRNA expression of Cyp1a2, Cyp2b10, Cyp3a11, Cyp4a10 and Cyp7a1, and corresponding nuclear receptors [aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-α (PPARα); farnesoid X receptor (FXR)]. In comparison, HgCl 2 and MeHg increased mRNA expression of Cyp1a2, Cyp2b10, Cyp4a10 and Cyp7a1 except for Cyp3a11, and corresponding nuclear receptors except for PXR. Western-blot confirmed mRNA results, showing increases in CYP1A2, CYP2B1, CYP2E1, CYP4A and CYP7A1 by HgCl 2 and MeHg only, and all treatments had no effects on CYP3A. Conclusions: Zuotai and Zuotai-containing 70W at clinical doses had minimal influence on hepatic CYPs and corresponding nuclear receptors, while HgCl 2 and MeHg produced significant effects. Thus, the use of total Hg content to evaluate the safety of HgS-containing 70W is inappropriate.

A novel naphthalimide that selectively targets breast cancer via the arylhydrocarbon receptor pathway

We report that the naphthalimide analogue 2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (NAP-6) is a highly potent and selective breast cancer targeting molecule. These effects are mediated via the aryl hydrocarbon receptor (AHR) pathway and the subsequent induction of CYP1 metabolising monooxygenases in breast cancer cell line models. Indeed the triple negative breast cancer cell line MDA-MB-468 with a GI50 value of 100 nM is greater than 500-fold more sensitive to NAP-6 compared with other tumour derived cell models. Within 1 h exposure of these cells to NAP-6, CYP1A1 expression increases 25-fold, rising to 250-fold by 24 h. A smaller concurrent increase in CYP1A2 and CYP1B1 is also observed. Within 24 h these cells present with DNA damage as evident by enhanced H2AXγ expression, cell cycle checkpoint activation via increased CHK2 expression, S-phase cell cycle arrest and cell death. Specific small molecule inhibitors of the AHR and CYP1 family ameliorate these events. A positive luciferase reporter assay for NAP-6 induced XRE binding further confirms the role of the AHR in this phenomenon. Non-sensitive cell lines fail to show these biological effects. For the first time we identify 2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione as a new AHR ligand that selectively targets breast cancer.

In vitro evaluation of the inhibition and induction potential of olaparib, a potent poly(ADP-ribose) polymerase inhibitor, on cytochrome P450

1. In vitro studies were conducted to evaluate potential inhibitory and inductive effects of the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, on cytochrome P450 (CYP) enzymes. Inhibitory effects were determined in human liver microsomes (HLM); inductive effects were evaluated in cultured human hepatocytes.2. Olaparib did not inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6 or CYP2E1 and caused slight inhibition of CYP2C9, CYP2C19 and CYP3A4/5 in HLM up to a concentration of 100 μM. However, olaparib (17–500 μM) inhibited CYP3A4/5 with an IC50 of 119 μM. In time-dependent CYP inhibition assays, olaparib (10 μM) had no effect against CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 and a minor effect against CYP3A4/5. In a further study, olaparib (2–200 μM) functioned as a time-dependent inhibitor of CYP3A4/5 (KI, 72.2 μM and Kinact, 0.0675 min−1). Assessment of the CYP induction potential of olaparib (0.061–44 μM) showed minor concentration-related increases in CYP1A2 and more marked increases in CYP2B6 and CYP3A4 mRNA, compared with positive control activity; however, no significant change in CYP3A4/5 enzyme activity was observed.3. Clinically significant drug–drug interactions due to olaparib inhibition or induction of hepatic or intestinal CYP3A4/5 cannot be excluded. It is recommended that olaparib is given with caution with narrow therapeutic range or sensitive CYP3A substrates, and that prescribers are aware that olaparib may reduce exposure to substrates of CYP2B6.

Delamanid does not inhibit or induce cytochrome p450 enzymes in vitro.

Delamanid is a new drug for the treatment of multidrug-resistant tuberculosis. Individuals who are co-infected with human immunodeficiency virus and Mycobacterium tuberculosis may require treatment with a number of medications that might interact significantly with the CYP enzyme system as inhibitors or inducers. It is therefore important to understand how drugs in development for the treatment of tuberculosis will affect CYP enzyme metabolism. The ability of delamanid to inhibit or induce CYP enzymes was investigated in vitro using human liver microsomes or human hepatocytes. Delamanid (100 µM) had little potential for mechanism-based inactivation on eight CYP isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Delamanid's metabolites were noted to inhibit the metabolism of some CYP isoforms, but these effects were observed only at metabolite concentrations that were well above those observed in human plasma during clinical trials. Delamanid (≤10 µM) did not induce CYP1A2, CYP2C9, and CYP3A4 activities in human hepatocytes, and there were no increases in CYP1A2, CYP2B6, CYP2C9, and CYP3A4 mRNA levels. Taken together, these data suggest that delamanid is unlikely to cause clinically relevant drug-drug interactions when co-administered with products that are metabolized by the CYP enzyme system.

Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes.2. Treatment of human hepatocytes for 72 h with 2–200 µM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 µM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 µM BHT.3. In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels.4. The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6β-hydroxylase activity.5. In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.

Hexachlorobenzene stimulates uroporphyria in low affinity AHR mice without increasing CYP1A2

Hexachlorobenzene (HCB), a weak ligand of the aryl hydrocarbon receptor (AHR), causes hepatic uroporphyrin (URO) accumulation (uroporphyria) in humans and animals. CYP1A2 has been shown to be necessary in the development of uroporphyria in mice. Using mice expressing the low affinity form of the AH receptor (AHRd), we investigated whether the enhancement of uroporphyria by HCB involves an obligatory increase in CYP1A2 as measured by specific enzyme assays and immunoblotting. We compared the ability of HCB, in combination with iron dextran and the porphyrin precursor, 5-aminolevulinate (ALA), to cause uroporphyria in a strain of mice (C57BL/6) which expresses the high affinity form of the receptor (AHRb 1), with three strains of mice (SWR and two 129 sublines) expressing the low affinity AHRd. In C57BL/6 mice, HCB-enhanced uroporphyria was associated with a doubling of CYP1A2. HCB treatment produced uroporphyria in iron-loaded mice expressing AHRd, even though there was little or no increase in CYP1A2. Cyp1a2(−/−) mice in a 129 background were completely resistant to HCB-induced uroporphyria, and female Hfe(−/−) 129 mice, in which the levels of hepatic CYP1A2 were half of those of the male levels, responded poorly. The effect of exogenous iron, administered in the form of iron dextran, on HCB enhancement of uroporphryia could be replicated utilizing the endogenous hepatic iron accumulated in 129 Hfe(−/−) mice. In conclusion, some minimal basal expression of CYP1A2 is essential for HCB-mediated enhancement of uroporphyria, but increases in CYP1A2 above that level are not essential.

Pharmacokinetics of intravenous theophylline in mutant Nagase analbuminemic rats

It was obtained from our laboratories that the expression of hepatic microsomal cytochrome P450 (CYP) 1A2 increased approximately 3.5 times in mutant Nagase analbuminemic rats (NARs, an animal model for human familial analbuminemia), and theophylline was reported to be metabolized to 1,3-dimethyluric acid (1,3-DMU) and 1-methylxanthine (which was further metabolized to 1-methyluric acid, 1-MU, via xanthine oxidase) via CYP1A2 in rats. Hence, the pharmacokinetic parameters of theophylline, 1,3-DMU and 1-MU were compared after intravenous administration of aminophylline, 5 mg/kg as theophylline, to control Sprague-Dawley rats and NARs. In NARs, the total area under the plasma concentration-time curve from time zero to time infinity (AUC) of theophylline was significantly smaller (1040 versus 1750 μg min/ml) than that in control rats and this could be due to significantly faster renal clearance (CL R, 1.39 versus 0.571 ml/min/kg, due to inhibition of renal reabsorption of unchanged theophylline) and nonrenal clearance (CL NR, 3.36 versus 2.25 ml/min/kg, due to 3.5-fold increase in CYP1A2) than those in control rats. Based on in vitro hepatic microsomal studies, the intrinsic 1,3-DMU formation clearance was significantly faster in NARs than that in control rats (267 versus 180 × 10 −6 ml/min). After intravenous administration of 1,3-DMU, the renal secretion of 1,3-DMU was inhibited in NARs. Inhibition of renal secretion or reabsorption of various compounds in NARs was also discussed.

Uroporphyria In Hfe Mutant Mice Given 5–Aminolevulinate: A New Model of Fe–Mediated Porphyria Cutanea Tarda

Porphyria cutanea tarda (PCT), a liver disease with skin lesions caused by excess liver production of uroporphyrin (URO), is associated with consumption of alcoholic beverages or estrogens, and moderate iron overload. Recently, it has been shown that many PCT patients carry mutations in the HFE gene, which is responsible for hereditary hemochromatosis. Mice homozygous for either the null mutation in the Hfe gene or the C282Y missense mutation rapidly accumulate hepatic parenchymal iron similar to patients with hemochromatosis. Here we investigated whether disruption of the murine Hfe gene would result in hepatic uroporphyria. Mice homozygous for the Hfe-null mutation accumulated high levels of hepatic URO when fed 5-aminolevulinate (ALA). Hfe (+/-) mice also accumulated hepatic URO when fed ALA, but at a much slower rate. The amount of accumulated URO in the null mutant mice was similar to that in wild-type mice treated with iron carbonyl in the diet, or injected with iron dextran. Iron in both wild-type and Hfe (+/-) mice was mostly in Kupffer cells. In contrast, Hfe (-/-) mice had considerable parenchymal iron deposition as well, in a pattern similar to that observed in wild-type mice treated with iron carbonyl. URO accumulation was accompanied by 84% and 33% decreases in hepatic uroporphyrinogen decarboxylase activities in Hfe (-/-) and Hfe (+/-) mice, respectively. No increases in CYP1A2 or other cytochrome P450s were detected in the Hfe-null mutant mice. We conclude that this experimental model of uroporphyria is a valid model for further investigations into the mechanism of PCT.

Role of small differences in CYP1A2 in the development of uroporphyria produced by iron and 5-aminolevulinate in C57BL/6 and SWR strains of mice

Previous work has implicated CYP1A2 in experimental uroporphyria caused by polyhalogenated aromatic compounds, and in uroporphyria caused by iron and 5-aminolevulinate (ALA) in the absence of inducers of CYP1A2. Here we examined whether the different susceptibilities of SWR and C57BL/6 strains of mice to uroporphyria in the absence of inducers of CYP1A2 are related to different levels of CYP1A2. Enzymological assays (ethoxy- and methoxyresorufin dealkylases, and uroporphyrinogen oxidation) and immunoblots indicated that there was about twice the amount of hepatic CYP1A2 in SWR mice compared with C57BL/6 mice. Immunohistochemistry revealed that CYP1A2 was located centrilobularly in the liver, and the staining was more intense in SWR mice than in C57BL/6 mice. Hepatic non-heme iron was about double in SWR compared with C57BL/6 mice. In SWR mice given iron dextran, hepatic iron was 1.7-fold that of C57BL/6 mice given iron dextran. SWR mice administered ALA in the drinking water accumulated much less hepatic protoporphyrin than did C57BL/6 mice. To confirm the importance of small increases in CYP1A2, C57BL/6 mice were given a low dose of 3-methylcholanthrene (MC) (15 mg/kg), as well as iron and ALA. There was about a 5- to 6-fold increase in hepatic uroporphyrin accumulation after 32 days on ALA compared with animals not given MC. In these animals, CYP1A2 was increased by 10-fold at 2 days, but returned to basal levels by 14 days. We conclude that small and transient differences in CYP1A2 may be important in the development of uroporphyria.

Modulation of hepatic cytochrome P450 activity and carcinogen bioactivation by black and decaffeinated black tea

The principal objective of this study was to compare the ability of green, black and decaffeinated black tea to modulate hepatic expression of cytochromes P450 in the rat, and the consequences on the bioactivation of some food-borne carcinogens. Furthermore, these studies allow inferences to be drawn as to the contribution of caffeine and flavanols in the tea-mediated changes in cytochrome P450 expression. Black tea is prepared from fresh tea leaf following oxidation of flavanols by polyphenol oxidases and consequently has a low content of these compounds. All three types of tea enhanced lauric acid hydroxylation but in the case of decaffeinated black tea no statistical significance was attained. Green tea and black tea, but not decaffeinated black tea, stimulated the O-dealkylations of methoxy-, ethoxy- and pentoxy-resorufin indicating upregulation of CYP1A and CYP2B. Immunoblot analysis revealed that green and black tea, but not decaffeinated black tea, elevated the hepatic CYP1A2 apoprotein levels. Hepatic microsomes from green and black tea-treated rats, but not those from the decaffeinated black tea-treated rats, were more effective than controls in converting IQ into mutagenic species in the Ames test. It is concluded that flavanols are not responsible for the effects of tea on the cytochrome P450 system, but caffeine could account for the increase in CYP1A2 and the consequent increase in the bioactivation of IQ.

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine.

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice.

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice. (Hepatology 1996 Mar;23(3):515-23)

Regenerative changes in hepatic morphology and enhanced expression of CYP2B10 and CYP3A during daily administration of cocaine

The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice. (Hepatology 1996 Mar;23(3):515-23)

Relative Sensitivities of 2,3,7,8-Tetrachlorodibenzo- p-dioxin-Induced Cyp1a-1 and Cyp1a-2 Gene Expression and Immunotoxicity in Female B6C3F1 Mice

Relative Sensitivities of 2,3,7,8 Tetracholorodibenzo- p-dioxin-Induced Cypla-1 and Cypla-2 Gene Expression and Immunotoxicity in Female B6C3F1 Mice. Narasimhan, T. R., Craig, A., Arellano, L., Harper, N., Howie, L., Menache, M., Birnbaum, L., and Safe, S. (1994). Fundam. Appl. Toxicol. 23, 598-607. Improvements in risk assessment require better linkage of exposure to response by the determination or target tissue dose. The relative sensitivity of several responses in female B6C3F1 mice was compared on the basis of administered and target tissue dose spanning 3 orders of magnitude. Twenty-four hours after administration, [ 3H]TCDD was detected in the heart, spleen, kidney, uterus, thymus, lung, and liver and the highest concentrations were noted in the liver, uterus, and lung. At doses from 5 to 25 ng/kg, hepatic [ 3H]TCDD levels associated with the cytosolic and nuclear subcellular fractions increased from 12 to 62% of the total liver levels and then decreased at higher doses. At the two lowest doses used in the enzyme induction study, 5 and 10 ng/kg, the levels of specifically bound nuclear Ah receptor complex liganded with [ 3H]TCDD were 2.3 and 2.5 fmol/mg protein. Slightly higher levels of nuclear Ah receptor complex were observed at doses between 25 and 100 ng/kg (i.e., 3.6 to 4.2 fmol/mg protein) and a steep dose-dependent increase in nuclear Ah receptor levels was noted at doses of 500, 1000, and 5000 ng/kg (8.0, 39.3, and 92.8 fmol/mg protein, respectively). The dose-dependent effects of [ 3H]TCDD on hepatic Cyp1a-1 and Cyp1a-2 mRNA levels, ethoxyresorufin O-deethylase (EROD) activity, and the splenic antibody plaque-forming cell (PFC) response to sheep red blood cells were also determined; the latter response was determined 9 days after administrations of TCDD. Statistically significant induction of hepatic Cyp1a-1 was observed at lower doses (25 ng/kg) than any other marker, followed by induction of EROD and PFCs expressed per spleen or per 10 6 cells which was observed at 100 ng TCDD/kg and at higher doses. Cyp1a-2 was elevated significantly relative to control at doses ⩾ 1000 ng/kg. The ED50 value for PFCs/106; cells was the lowest of the variables analyzed and was not statistically significantly different from control (91 ± 92 ng/kg). A 500% increase in Cyp1a-2 and Cyp1a-1 mRNA levels was observed at doses of 736 ± 132 and 1630 ± 431 ng/kg, respectively. Due to variability in response in PFCs/spleen and the submaximal induction of EROD activity, ED50 values could not be calculated for these responses. The analyses indicate that the immunosuppressive response (when normalized for the number of spleen cells) may be depressed by administered doses as low as 90 ng TCDD/kg body weight. A 50% in crease in Cyp1a-1 or Cyp1a-2 was observed at higher administered doses (1630 or 736 ng/kg, respectively). This suggests that the immunosuppressive response is depressed at lower doses of TCDD than the other variables studied.

Association between growth stimulation by phenobarbital and expression of cytochromes P450 1A1, 1A2, 2B1/2 and 3A1 in hepatic hyperplastic nodules in male F344 rats.

This study was conducted to examine relationships between phenobarbital (PB) treatment, specific cytochrome P450 gene expression patterns and growth rates of hepatic hyperplastic nodules. Nodules were induced in 8 week old male F344 rats by a Solt-Farber resistance protocol. Six weeks after diethylnitrosamine (DEN) initiation, subgroups of rats were either kept on control chow diet or transferred to a chow diet containing 0.05% PB, then killed 2 weeks later. [3H]Thymidine was delivered continuously via osmotic minipump during the final 3 days of the experimental to label dividing cells. PB treatment resulted in a 89% increase in the number of persistent gamma-glutamyl transpeptidase (GTT) nodules per cm2 section, a 278% increase in the area of persistent GGT nodules per cm2 section, and a 116% increase in the average area per persistent nodule. PB increased the number of [3H]thymidine-labeled persistent GGT nodules but did not significantly change the labeling index (LI) distribution pattern or the average LI. A slight but uniform increase in CYP1A2 expression (relative to surrounding, non-nodular tissue) was observed in 50% (23/46) and 59% (60/102) of persistent nodules in control and PB-treated animals respectively. In contrast, for nodules undergoing remodeling, CYP1A2 expression was elevated in only 9% (2/22) and 0% (0/24) in control and PB groups respectively. In the PB group, CYP2B1/2 was underexpressed in 53% (54/102) of persistent GGT nodules and in 0% (0/24) of the remodeling nodules. Comparing LI among the persistent GGT nodules, those that displayed simultaneous increases in CYP1A2 and decreases in CYP2B1/2 had the highest LI, and were followed in level by those expressing either increases in CYP1A2 or decreases in CYP2B1/2. Nodules that expressed both CYP1A2 and 2B1/2 in a manner similar to the surrounding tissue had the lowest LI. Thus, these data suggest that expression of specific forms of cytochrome P450 may be an important factor in determining other phenotypic characteristics, e.g. rate of cell proliferation and GGT expression, within specific nodules.