Validation of Precision-Cut Liver Slices in Dynamic Organ Culture as anin VitroModel for Studying CYP1A1 and CYP1A2 Induction

doi:10.1006/taap.1996.0236

Toxicology and Applied Pharmacology

Volume 140, Issue 2, October 1996, Pages 393-403

https://doi.org/10.1006/taap.1996.0236 Get rights and content

Abstract

The utilization of precision-cut liver slices in dynamic organ culture as anin vitromodel was validated by comparing the induction of the biomarker responses followingin vitro(rat liver slice) andin vivoexposure of rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The biomarker responses investigated were cytochrome P450s 1A1 and 1A2 (CYP1A1 and CYP1A2) mRNA, protein, and activities. Precision-cut rat liver slices were incubated in dynamic organ culture for 24 hr with medium containing 0.001–10 nMTCDD or medium without TCDD (control). The resultant mean TCDD concentration in the slices ranged from 19 to 80,925 ppt (wet wt), respectively. A concentration-dependent induction of CYP1A1 mRNA, protein, and activities and a more modest induction of CYP1A2 mRNA was observed in liver slices at all medium concentrations of TCDD. The O-demethylation of 7-methoxyresorufin, a marker for CYP1A2 activity, was induced at TCDD medium levels of 0.01 nMand greater, whereas a detectable increase in CYP1A2 protein occurred only at the higher concentrations. Comparable liver concentrations of TCDD (8–64,698 ppt wet wt) were achieved at 24 hr following a singlein vivoexposure of rats to TCDD at doses ranging from 0.002 to 5 μg/kg po. Concentration–effect and dose–response relationships for induction of CYP1A1 and CYP1A2 were similar followingin vitroandin vivoexposure to TCDD, although the magnitude of induction was greater forin vivoexposure. The data support the use of liver slices in dynamic organ culture for assessing the relativein vivopotency of a compound to induce CYP1A1 and CYP1A2. Human tissue can also be readily utilized in thisin vitromodel to predict the biological and toxicological effects of a givenin vivoexposure to TCDD.

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A similar slight induction has been noted for cynomolgus CYP1A2 (Edwards et al., 1994). Similarly, CYP1A1 mRNA and protein levels increased in human liver slices treated with TCDD, resulting in increased enzyme activity; in contrast, the induction of CYP1A2 mRNA was modest (Drahushuk et al., 1996). In marmosets and cynomolgus macaques, CYP1A2 mRNA is induced by 3-methylcholanthrene (Sakuma et al., 1997, 1998), similar to human CYP1A2 in primary hepatocytes (Rhodes, Otten, Hingorani, Hartley, & Franklin, 2011).
Cynomolgus macaques (Macaca fascicularis, an Old World monkey) are widely used in drug development because of their genetic and physiological similarities to humans, and this trend has continued with the use of common marmosets (Callithrix jacchus, a New World monkey). Information on the major drug-metabolizing cytochrome P450 (CYP, P450) enzymes of these primate species indicates that multiple forms of their P450 enzymes have generally similar substrate selectivities to those of human P450 enzymes; however, some differences in isoform, activity, and substrate specificity account for limited species differences in drug oxidative metabolism. This review provides information on the P450 enzymes of cynomolgus macaques and marmosets, including cDNA, tissue expression, substrate specificity, and genetic variants, along with age differences and induction. Typical examples of important P450s to be considered in drug metabolism studies include cynomolgus CYP2C19, which is expressed abundantly in liver and metabolizes numerous drugs. Moreover, genetic variants of cynomolgus CYP2C19 affect the individual pharmacokinetic data of drugs such as R-warfarin. These findings provide a foundation for understanding each P450 enzyme and the individual pharmacokinetic and toxicological results in cynomolgus macaques and marmosets as preclinical models. In addition, the effects of induction on some drug clearances mediated by P450 enzymes are also described. In summary, this review describes genetic and acquired individual differences in cynomolgus and marmoset P450 enzymes involved in drug oxidation that may be associated with pharmacological and/or toxicological effects.
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Similarly, the treatment of human liver slices with TCDD in dynamic organ culture increases the levels of P450 1A1 mRNA and the protein and, consequently, results in increased enzyme activity. At the same time, the induction of P450 1A2 mRNA in liver slices was more modest [26]. In marmosets and cynomolgus macaques, P450 1A2 mRNA is commonly induced by 3-methylcholanthrene [21,23]; similar results were obtained for P450 1A2 in primary human hepatocytes [27].
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Citation Excerpt :
In this study, the EC50 for hepatic CYP1A1 activity for TCDD was approximately 5 ng/g liver. Other studies in rats and mice, including SYSTEQ data, have reported EC50 values in the same range for induction of hepatic CYP1A1 by TCDD, irrespective of the administration protocol (DeVito et al., 1997; Diliberto et al., 2001; Drahushuk et al., 1996; van Birgelen et al., 1995a; Vanden Heuvel et al., 1994). Thus, regardless of the use of a single or multiple oral dosages, it is shown that EC50 values for induction of hepatic CYP1A1 by TCDD are comparable if based on tissue concentrations.
Relative effect potencies (REPs) for dioxins and dioxin-like compounds based on tissue concentration or internal dose (^systemicREPs) can be considered of high relevance for human risk assessment. Within the EU-project SYSTEQ, ^systemicREPs for 1,2,3,7,8-pentachlorodibenzodioxin (PeCDD), 2,3,4,7,8,-pentachlorodibenzofuran (4-PeCDF), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118) and 2,3,3′,4,4′,5-hexachlorobiphenyl (PCB 156) were calculated based on a plasma, adipose tissue or liver concentration in Sprague Dawley rats and C57bl/6 mice three days after a single oral dose. Compound-specific distribution as well as differences in accumulation and elimination can influence the tissue concentration and thereby the relative potency estimate of a congener. Here, we show that distribution patterns are generally similar for the tested congeners between the SYSTEQ dataset and other studies using either a single dose or subchronic dosing. Furthermore, the responding concentration for TCDD in single dose studies is comparable to the responding concentrations reported in subchronic studies. In contrast with data for laboratory rodents, available distribution data for humans in the general population display little or no hepatic sequestration. Because hepatic sequestration due to CYP1A2 protein binding may affect the amount of congener that is bioavailable for the AhR to produce hepatic responses, estimates of relative potencies between congeners with differing degrees of hepatic sequestration based on hepatic responses may be misleading for application to human risk assessment. Therefore, extra-hepatic concentration in blood serum/plasma or adipose tissue together with a biological extra-hepatic response might give a more accurate prediction of the relative potency of a congener for human responses under environmental conditions.
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2012, Toxicology
Citation Excerpt :
Samples were stored at −20 °C. Activation of the AhR was estimated through measurement of the induction of CYP1A1 mRNA (Drahushuk et al., 1996; Gaylor and Aylward, 2004; Vanden Heuvel et al., 1994) in rat H4IIE cells and human MCF-7 cells using quantitative real-time PCR (qRT-PCR). Further analysis of CYP1B1 and CYP1A2 was conducted in rat H4IIE cells.
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Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters retinoid homeostasis in maternal and perinatal tissues of the Holtzman rat
2007, Toxicology and Applied Pharmacology
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), one of the most widely studied environmental contaminants, causes a variety of adverse health effects including teratogenesis and altered development which may be related to disruptions in retinoid homeostasis. The purpose of this study was to determine the effect that gestational administration of TCDD has on retinoid homeostasis in both pregnant Holtzman rats and developing fetuses and neonates. A single oral dose of TCDD (0, 1.5, 3, or 6 μg/kg) was administered to pregnant rats on gestation day 10, with fetuses analyzed on gestation days 17 and 20, and neonates analyzed on post natal day 7. Exposure to TCDD generally produced decreases in the concentrations of retinyl esters, such as retinyl palmitate, and retinol in maternal and perinatal liver and lung, while increasing levels in the maternal kidney. Additionally, perinatal hepatic retinol binding protein 1-dependent retinyl ester hydrolysis was also decrease by TCDD. Sensitivity of the developing perinates to TCDD appeared to have an age-related component demonstrated by an increased rate of mortality and significant alterations to body weight and length on post natal day 7 relative to that observed at gestation day 20. A unique observation made in this study was a significant decrease in lung weight observed in the perinates exposed to TCDD. Taken together, these data demonstrate that TCDD significantly alters retinoid homeostasis in tissues of the developing fetus and neonate, suggesting that their unique sensitivity to TCDD may at least be in part the result of altered retinoid homeostasis.

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^☆: Supported by NIEHS Grant R01 ES06556-01.

^☆☆: Presented in part at the Annual Meeting of the Society of Toxicology (Toxicologist15,63, 1995).

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Regular ArticleValidation of Precision-Cut Liver Slices in Dynamic Organ Culture as anin VitroModel for Studying CYP1A1 and CYP1A2 Induction☆,☆☆

Abstract

Regular Article
Validation of Precision-Cut Liver Slices in Dynamic Organ Culture as anin VitroModel for Studying CYP1A1 and CYP1A2 Induction☆,☆☆