Induction of a pleiotropic response by phenobarbital and related compounds: Response in various inbred strains of rats, response in various species and the induction of aldehyde dehydrogenase in copenhagen rats

doi:10.1016/0006-2952(92)90483-Y

Biochemical Pharmacology

Volume 44, Issue 8, 20 October 1992, Pages 1651-1660

https://doi.org/10.1016/0006-2952(92)90483-Y Get rights and content

Abstract

The ability of phenobarbital (PB) to induce a “pleiotropic response” which includes both cytochromes P450 (CYP) as well as other drug-metabolizing enzymes was investigated in mice, rabbits, hamsters, and various inbred strains of rats. PB induced similar drug-metabolizing enzymes (CYP2B, CYP3A, and epoxide hydrolase) in rats, mice, rabbits and hamsters. PB and two structural analogues (ethylphenylhydantoin and barbital) induced a variety of drug-metabolizing enzymes (CYP2B, CYP3A, CYP2A, epoxide hydrolase) in a series of inbred strains of rats. In contrast, levels of aldehyde dehydrogenase (ALDH) (propionaldehyde, NAD⁺) which were expressed constitutively in all strains of rats were induced by PB in only two of the eight strains (ACI, Copenhagen). Further investigations of ALDH induction by structurally diverse compounds in Copenhagen rats demonstrated a strong correlation between the induction of ALDH and other elements of the pleiotropic response (CYP2B, CYP3A, epoxide hydrolase). These results imply that induction of ALDH (propionaldehyde, NAD⁺) is associated with the PB pleiotropic response in Copenhagen rats.

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Each assay was conducted in triplicate. Aldehyde dehydrogenase (ALDH) activities were determined in the cytosolic fractions according to Jones and Lubet (1992) using propanal as substrate. The assay closely resembled that for ADH, measuring the formation of NADH by spectrophotometry in transparent 96-well plates at 37 °C.
In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To determine their relative contribution, these activities were compared to those of untreated adult male rat liver, using commonly accepted assays. The enzymes comprised cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), esterase, UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). In contrast to liver, no activities were measurable for 7-ethylresorufin-O-dealkylase (CYP1A), 7-pentylresorufin-O-dealkylase (CYP2B), 7-benzylresorufin-O-dealkylase (CYP2B, 2C and 3 A), UGT1, UGT2 and GST in placenta, indicating that the placental activity of these enzymes was well below their hepatic activity. Low activities in placenta were determined for FMO (4%), and esterase (8%), whereas the activity of placental ADH and ALDH accounted for 35% and 40% of the hepatic activities, respectively. In support of the negligible placental CYP activity, testosterone and six model azole fungicides, which were readily metabolized by rat hepatic microsomes, failed to exhibit any metabolic turnover with rat placental microsomes. Hence, with the possible exception of ADH and ALDH, the activities of xenobiotic-metabolizing enzymes in rat placenta are too low to warrant consideration in PBTK modelling.
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Alcohol dehydrogenase (ADH; EC. 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3) play important roles in the metabolism of both endogenous and exogenous alcohols and aldehydes. The expression and localisation patterns of ADH (1–3) and ALDH (1–3) were investigated in the skin and liver of the mouse (BALB/c and CBA/ca), rat (F344) and guinea-pig (Dunkin-Hartley), using Western blot analysis and immunohistochemistry with class-specific antisera. ALDH2 expression and localisation was also determined in human skin, while ethanol oxidation, catalysed by ADH, was investigated in the mouse, guinea-pig and human skin cytosol. Western blot analysis revealed that ADH1, ADH3, ALDH1 and ALDH2 were expressed, constitutively, in the skin and liver of the mouse, rat and guinea-pig. ADH2 was not detected in the skin of any rodent species/strain, but was present in all rodent livers. ALDH3 was expressed, constitutively, in the skin of both strains of mouse and rat, but was not detected in guinea-pig skin and was absent in all livers. Immunohistochemistry showed similar patterns of expression for ADH and ALDH in both strains of mouse, rat, guinea-pig and human skin sections, with localisation predominantly in the epidermis, sebaceous glands and hair follicles. ADH activity (apparent V_max, nmoles/mg protein/min) was higher in liver (6.02–16.67) compared to skin (0.32–1.21) and lower in human skin (0.32–0.41) compared to mouse skin (1.07–1.21). The ADH inhibitor 4-methyl pyrazole (4-MP) reduced ethanol oxidation in the skin and liver in a concentration dependent manner: activity was reduced to ≈30–40% and ≈2–10% of the control activity, in the skin and liver, respectively, using 1 mM 4-MP. The class-specific expression of ADH and ALDH enzymes, in the skin and liver and their variation between species, may have toxicological significance, with respect to the metabolism of endogenous and xenobiotic alcohols and aldehydes.
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The induction of a hepatic pleiotropic response, including increase in liver/body weight ratio, induction of hepatic CYP2B and CYP3A protein and catalytic activity, and hepatic microsomal epoxide hydration activity, was investigated in male cotton rats (Sigmodon hispidus) administered graded dietary concentrations (0–1500 ppm) of phenobarbital (PB) for 14 days. A dose-dependent induction of each endpoint was observed, although plateaus in the various dose-response curves were not obtained, and ED₅₀ values (PB concentrations associated with half-maximal responses) for the various endpoints were not able to be calculated. A maximal 1.31-fold increase, compared to the control value, in liver/body weight ratio was observed, while microsomal epoxide hydration activity was increased as much as 3.6-fold by PB administration. Pentoxy- and benzyloxyresorufin O-dealkylation and testosterone 16β-hydroxylation activities (considered to be relatively selective for CYP2B in the Norway rat (Rattus norvegicus)), were induced maximally less than five-fold. Testosterone 6β-hydroxylation (considered to be relatively selective for CYP3A in R. norvegicus) was induced maximally less than two-fold. Maximal induction of 7-ethoxy-4-trifluoromethyl-coumarin O-deethylation was 18-fold, compared to the control rate. Western blotting studies indicated that hepatic microsomal proteins immunoreactive with polyclonal antisera to R. norvegicus CYP2B1 or CYP3A1 were induced, in a dose-responsive manner, by PB in the cotton rats. These results indicate that the cotton rat responds to PB treatment with a coordinate pleiotropic response similar to that displayed by R. norvegicus, although the substrate specificity of the induced proteins appears to differ between the two rodent species.
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Induction of a pleiotropic response by phenobarbital and related compounds: Response in various inbred strains of rats, response in various species and the induction of aldehyde dehydrogenase in copenhagen rats

Abstract

Biochem Pharmacol

Biochem Pharmacol

Arch Biochem Biophys

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Chem Biol Interact

Anal Biochem

J Biol Chem

Arch Biochem Biophys

Arch Biochem Biophys

J Biol Chem

J Biol Chem

J Biol Chem

Biochem Pharmacol

J Biol Chem

J Biol Chem

J Biol Chem

Genomics

Hemin administration to rats reduces levels of hepatic mRNAs for phenobarbitone-inducible enzymes

Mol Pharmacol

Differentiated induction of cytochrome P450b/e and P450p mRNAs by dose of phenobarbital in primary cultures of adult rat hcpatocytes

Mol Pharmacol