Comparison of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced CYP1A1 gene expression profile in lymphocytes from mice, rats, and humans: Most potent induction in humans

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exerts its toxicity by binding a transcription factor, the aryl hydrocarbon receptor (AhR). C57BL/6 (C57) mice express AhRs that have high affinity for TCDD, and they strongly express target genes and develop severe toxic effects upon TCDD exposure. By contrast, DBA/2 (DBA) mice have a low-affinity form of AhR, weakly express target genes, and are resistant to TCDD. Although humans express low-affinity AhRs and have been assumed to be refractory to TCDD, their sensitivity to TCDD has yet to be determined. In this study we compared the TCDD-induced CYP1A1 gene expression profiles in lymphocytes from humans, C57 mice, DBA mice, and SD rats to obtain data as a basis for estimating human sensitivity to TCDD. Lymphocyte fractions prepared from the blood of individual humans and animals were cultured with TCDD. Their mRNAs for CYP1A1 and housekeeping genes were measured by RT-PCR or real-time PCR with primers designed for regions that are 100% homologous among each of the genes of all species/strains tested to obtain similar PCR efficiency. TCDD-induced CYP1A1 expression peaked at 2 h in DBA mice and SD rats and at 6 h in C57 mice and humans. At the peak times human lymphocytes showed the most potent CYP1A1 mRNA induction of the four species/strains tested. These results suggest that human lymphocytes are more sensitive to TCDD than the lymphocytes of mice and rats. Since the AhR-dependent gene expression did not reflect the AhR affinity for TCDD, these results also suggest that AhR-dependent gene expression in lymphocytes is modulated by an as yet unidentified mechanism in addition to the AhR affinity.

Introduction

Dioxins, including the most potent congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are widespread environmental contaminants that exert adverse health effects by binding a transcription factor, the aryl hydrocarbon receptor (AhR) (Schmidt and Bradfield, 1996, Fujii-Kuriyama and Mimura, 2005). Upon binding to ligands, the AhR becomes activated and dimerizes with another transcription factor, the aryl hydrocarbon receptor nuclear translocator (ARNT). The AhR/ARNT heterodimer specifically binds DNA sequences called xenobiotic responsive elements (XREs) that are distributed in the enhancer regions of various genes, including one of the most sensitive targets, the CYP1A1 gene, and up-regulates or modulates their expression. These changes in gene expression are thought to cause a variety of toxic effects.

Although the AhR-dependent pathway is conserved and leads to toxic effects of TCDD in many mammalian species, susceptibility to the toxicity of TCDD varies greatly among animal species and strains (Schmidt and Bradfield, 1996, Simanainen et al., 2004, Karchner et al., 2006). This is one of the findings that have aroused concern about human susceptibility to TCDD toxicity. The primary cause of species- and strain-specific sensitivity has been attributed to the affinity of the AhR for ligands, as shown by the precise studies in mouse strains. Mice of the “sensitive” strain C57BL/6 (C57) express an AhR with high affinity for ligands, and mice of the “insensitive” strain DBA/2 (DBA) express a low-affinity AhR. The dissociation constant (K_d) of the AhR of C57 and DBA mice for TCDD has been reported to be 1.8 and 16 nM, respectively, by Okey et al. (1989) and 0.27 and 1.66 nM, respectively, by Ema et al. (1994). The difference between their affinities correlates well with the strain-specific difference in AhR-dependent gene expression and sensitivity to TCDD toxicity between C57 mice and DBA mice. CYP1A1 and CYP1B1 are more strongly induced in C57 mice by TCDD exposure than in DBA mice (Abel et al., 1996). CYP1A1-mediated ethoxyresorufin O-deethylase (EROD) activity in the liver was induced at an ED50 of 1.1 and 16 μg/kg in C57 mice and DBA mice, respectively, and hepatomegaly developed at doses of 3 μg/kg or higher and 97.5 μg/kg or higher, respectively (Weber et al., 1995).

The human AhR structurally resembles the DBA AhR more closely than the C57 AhR (Dolwick et al., 1993, Ema et al., 1994), and the K_d for TCDD (1.58 nM) is just slightly lower than that of the DBA AhR (Ema et al., 1994). Human AhR knock-in mice exhibit hepatic CYP1A1 induction by AhR ligands that is comparable to or less than the level of induction in DBA mice (Moriguchi et al., 2003). These findings suggest that humans are “resistant” to TCDD. However, AhR function may be affected by a variety of factors, such as coactivators, other transcription factors, and chaperone proteins (Carlson and Perdew, 2002, Fujii-Kuriyama and Mimura, 2005, Hankinson, 2005). These factors may explain the cell-type-specific differences in TCDD-induced responses that do not simply depend on the nature of the AhR of individual species/strains.

Previous studies compared TCDD-induced EROD activity and/or CYP1A1 induction in primary hepatocytes and embryonic palate organ cultures from humans and animals (Abbott et al., 1999, Silkworth et al., 2005, Xu et al., 2000). Although these studies in primary cells or tissues have been very informative in terms of understanding the vulnerability of humans and animals to TCDD, comparative studies of AhR activity in other types of primary cells have been very limited. Lymphocytes are readily accessible primary cells that are available to examine the sensitivity to AhR-ligands. While lymphocytes are known to be one of the primary targets of TCDD immunotoxicity (Kerkvliet, 2002) and human lymphocytes have been used in numerous studies to investigate the role of CYP1A1-mediated enzyme activity in chemical carcinogenesis (Nebert et al., 2004), there have been no studies comparing AhR function in lymphocytes from humans and various animal species. A comparison of responses to TCDD by primary lymphocytes from humans and animals that have been used in immunotoxicity studies would provide a basis for estimating the susceptibility of the human immune system to TCDD.

In the present study we investigated TCDD-induced gene expression profiles in lymphocytes freshly prepared from blood of healthy human individuals, C57 mice, and DBA mice, using CYP1A1 gene as a marker gene. We also compared the sensitivity of SD rats, which have a high affinity AhR for TCDD (K_d, 0.2–0.5 nM) (Denison et al., 1986, Rannug et al., 1987, Sloop and Lucier, 1987) and are known to be another TCDD-sensitive strain. To directly compare the induction of CYP1A1 and housekeeping genes in different species/strains, we established PCR primers for individual genes that have similar PCR efficiency in all the species tested.