Mechanistic and molecular studies into the effects of 2,3,7,8-tetrachlorodibenzo-P-dioxin and similar compounds in the deer mouse, Peromyscus maniculatus



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Texas Tech University


Much work has been done with environmental endocrine disrupters in laboratory animal models; however, much work remains to be done in wildlife species used as sentinels. In this study, the effects of 2,3,7,8-TCDD, B[a]P, 3-MC, PB and p,p'-DDE were characterized in the deer mouse. Deer mice were dosed by gavage with these chemicals and morphological endpoints of brain- somatic index, liver-somatic index and gonadal-somatic index measured. In addition, enzyme induction and expression were measured for comparison with other species. In contrast to common laboratory rodents, deer mice were shown to have a distinct dioxin-like response to p'p-DDE, specifically the induction of cytochrome P4501A1.

Risk is a function of toxicity, physiology and exposure. It is important to understand all of these factors to gain a good understanding of the risk posed by a certain toxicant. As such, this study also addresses bioavailability of one such toxicant, p,p'-DDE, in soil. Soil was compounded with varying concentrations (0, 10, 200 PPM) of p,p'-DDE and deer mice were exposed to the soil for varying amounts of time (1, 2, or 3 weeks) . The same endpoints were measured as in the first phase of the study. Though gas chromatography analysis of tissue samples indicated that p,p'-DDE was entering and accumulating in the livers of deer mice, no overt signs of toxicity were observed other than a significant decrease in gonadal-somatic and uterine wet weight indices between control and high dose groups in mice exposed to contaminated soils for one week, and a significant increase in liver somatic indices between control and high dose groups for mice exposed to contaminated soils for two weeks.

2,3,7,8-TCDD exerts its effects through binding with the Ah receptor and ARNT and the resultant binding of that complex to DNA. 2,3,7,8-TCDD binds to the Ah receptor at the ligand-binding domain located within the PAS domain. Apart from the AhR and ARNT, other important proteins also contain the conserved PAS domain, namely the clock-associated proteins PER and BMAll. PER is important in the maintenance of circadian rhythms. This study therefore also touches on the possible effects that 2,3,7,8-TCDD has on circadian rhythm, effects that have far-reaching implications. 2,3,7,8-TCDD was found to advance the on-set of activity in deer mice by 3 hours, making the mice active during part of the light-dark cycle when they normally sleep. Also, the clock-associated proteins mPERl and mBMALl were quantitated through Western blotting. Results indicate that 2,3,7,8-TCDD administered at ZT18 phase delays the circadian rhythm of mPERl while also decreasing the expression of both mPERl and mBMALl compared to controls.

One explanation for the induction of CYPlAl in the deer mouse and not in the laboratory mouse may lie in differences in the AhR and ARNT. Therefore, the full-length Ah receptor and ARNT cDNA were cloned and sequenced in order that a comparison could be made with the known sequences in other species. Molecular analysis of the Ah receptor gene in the deer mouse is an essential step in attempts to understand differences in Ah receptor between species. ARNT itself is also very important, as it is the common factor in three environmental sensing pathways: 2,3,7,8-TCDD response, hypoxia response, and circadian response. As such, it provides a means by which the three pathways can interact. We have compared the published sequences for other species to that of the deer mouse Ah receptor and ARNT. For the Ah receptor, we had hypothesized that differences would lie in the ligand-binding domain. Deer mice AhR sequence data indicate a potentially significant amino acid substitution in position 236 (valine for methionine). The side chain differences could influence protein folding, thereby altering the structure of the protein and possibly access to the critical ligand-binding site. The deer mouse 7VNRT was found to have a high degree of homology with the house mouse and rat ARNTs, especially in the critical domains - the bHLH, PAS A, PAS B and transcription activation domains. However, amino acid substitutions in the transcription activation domain are such that there could be differences in ARNT function amongst the three species. It is crucial to understand the role of receptor polymorphisms and the mechanisms that underlie the toxicity to environmental contaminants such as 2,3,7,8-TCDD in this species in order to compare risk of exposure across species.



Dioxins -- Physiological effect, Peromyscus maniculatus, Peromyscus maniculatus -- Genetics