I think it is very difficult to give a satisfying answer to your question as PAH effects are dependent on the amounts taken up and the CYP activities which may not be necessarily zero with respect to PAH oxidation. If there is a low level of PAH oxidative metabolism (either due to low uptake or low enzymatic activity) there is good chance that the reactive metabolites are effciently 'neutralized' by phase II reactions. This means that there should be a favourable balance between PAH activation, deactivation and excretion. I think that enzymes of the phase II system are not suppressed by estradiol in fish (?) otherwise the activated PAHs become able to react with DNA or other cellular structures.

Another aspect: estradiol itself may become a carcinogen through the action of CYP1 enzymes by formation of catecholestrogens. If estradiol suppresses EROD it concomitantly suppresses its possible own transformation to catecholestrogens.

The answer to your question cannot be given in general as the output of PAH effects depends on the dosis, the CYP-activities and the phase II activities. Importantly, also the types of ingested PAH / the composition of the PAH mixture plays a role as the different PAHs exhibit different carcinogenic potential if at all.

Peter

Thank you for such a thoughtful reply. I was perhaps expecting too much that there might be an easy answer! In the paper* that I was quoting from, EROD activity (in response to a PAH) was suppressed at an even lower dose of estradiol than that required to induce vitellogenin production (repeatable result). Also, whereas ethinyl estradiol was more potent than E2 in inducing vitellogenin production, the reverse was the case for suppression of phase I metabolism (i.e. it appeared to have high specificity for estradiol). This suggests (though of course does not prove) that this is an evolved function of estradiol (rather than some non-specific effect). The next step in the hypothesis is that a specific function would only have evolved if it provided a selective advantage for the females.

For everyone reading this post, I apologise for referring to 'polyaromatic hydrocarbons' instead of 'polycyclic aromatic hydrocarbons'

*Kirby, M. F.; Smith, A. J.; Neall, P.; Rooke, J.; Scott, A. P.; Katsiadaki, I. Ethoxyresorufin-O-deethylase (EROD) and vitellogenin in flounder (Platichthys flesus): System interaction, crosstalk and implications for monitoring. Aquat. Toxicol. 2007, 81, 233-244.

If you need a copy of this paper, send me a request. Copyright laws mean that I am not allowed to attach it to a general post.

Hi Alexander,

Before addressing whether it would be good or bad for a fish having both estradiol and PAHs/dioxin, lets turn first to the facts. Those presented in your question obviously refer to the data of 2001-2003 (and the paper of Kurby of 2007), where rat liver hepatoma H4IIE and rainbow trout hepatocytes, while treated with high (non-physiological) concentrations of estradiol (except that in paper of Kurby), all showed the TCDD-induced EROD activity decreased. The authors suggested estradiol-mediated suppression did not rely on the synthesis of protein factors but rather might hinder the formation of the activated TCDD/AhR complex and so the subsequent binding on DRE.

However, followed studies revealed much more complicated picture. Thus in 2010, Grans et al. published a paper “One-way inhibiting cross-talk between arylhydrocarbon receptor (AhR) and estrogen receptor (ER) signaling in primary cultures of rainbow trout hepatocytes”. Aquat. Toxicol. 100, 263–270 (2010). They investigated cross-talk between ER-VTG and AhR-CYP1A signaling pathways, using quantitative PCR (qPCR) for mRNA analyses and studies of CYP1A catalytic function and protein expression. Combined exposure to BNF and EE(2) reduced the EE(2)-mediated induction of VTG mRNA levels by about 40%, but had no effect on the BNF-mediated CYP1A mRNA levels, indicative of a one-way inhibiting AhR-ER cross-talk. Moreover, in the lab. of S. Kato and Y. Fujii-Kawajiri, it was shown that cross-talk of dioxins with estrogen and androgen signaling pathways that modulation of estrogen/androgen signaling by dioxins, and is exerted in part through direct association of AhR with estrogen (ER) or androgen (AR) receptors. Those findings revealed the roles of the ubiquitin system in sensing and biological response to environmental chemicals, in which AhR acts as a ubiquitin ligase component to enhance the destruction of specific substrates (J Steroid Biochem Mol Biol.127(1-2):102-7, 2011).

Just during last year, two papers were published justifying the above mechanisms. First, by L. Poellinger et al entitled “A Molecular Mechanism To Switch the Aryl Hydrocarbon Receptor from a Transcription Factor to an E3 Ubiquitin Ligase”, where the TCDD/PAHs Ah receptor was identified the molecular switcher that determined the receptor as a transcription factor or an E3 ubiquitin ligase (Mol Cell Biol.;37(13) 2017). They used the breast cancer cell line MCF7, which expresses a functional estrogen receptor alpha (ERα) signaling pathway, suggested that aryl hydrocarbon receptor nuclear translocator (ARNT) plays an important role in the modulation of the dual functions of the AhR. They also used the MCF7 cells overexpressing the AhRR showed lower ERα protein levels, reduced responsiveness to estradiol, and reduced growth rates. Importantly, when these cells were used to produce estrogen-dependent xenograft tumors in SCID mice, observed lower ERα protein levels and a reduced tumor mass was observed. The second paper’s object of study fitted directly to your point, namely primary rainbow trout hepatocytes. M. Hultman et al paper is entitled “Characterizing combined effects of antiestrogenic chemicals on vitellogenin production in rainbow trout (Oncorhynchus mykiss) hepatocytes”. The authors wrote: Fish are exposed to a complex mixture of endocrine disrupting compounds (EDC), some of which display antiestrogenic activity leading to suppression of estrogen receptor (ER)- mediated reproductive processes. The aim of the present study was to characterize single and combined antiestrogenic effects of the aryl hydrocarbon receptor (AhR)-agonist β-naphthoflavone (BNF) and ER-antagonist 4-hydroxytamoxifen (OHT) on vitellogenin (Vtg) protein using primary rainbow trout (Oncorhynchus mykiss) hepatocytes. Data demonstrated that both BNF and OHT significantly reduced 17β-estradiol (E2)-induced Vtg protein expression in a concentration responsive manner, whereas exposure to a mixture of these produced an additive antiestrogenic effect. The results observed at the protein level were further supported by transcriptional analysis of ER-responsive genes (er-α, vtg-1, zrp), where only E2-induced vtg-1 gene expression was significantly decreased by OHT and the mixture of OHT and BNF. E2-induced er-α and zrp gene expression was not markedly altered. The significant reduction of E2-induced vtg-1 gene expression by OHT suggested that the antiestrogenic effect of this compound may be associated with ER signaling pathway. Specific genes involved in putative AhR-ER cross-talk were also investigated, however none were directly associated with the compound anti-estrogenic mode of action. Although this action of the single compounds and mixture were not completely characterized, the present study enhanced our knowledge of the combined toxicity. (J Toxicol Environ Health A. 2017;80(16-18):987-1001).

Returning to your question “is this a good or a bad thing for the animal” of being under influence of both estradiol and PAHs/dioxin, nowadays the real stress of environmental pollutants like PAHs and dioxins on estradiol producing fish population might be almost quantitatively assessed by animal’ vital parameters.

Best wishes,

Ilya

Thankyou for your answer Ilya. Sorry for the delay in replying. I interpret from what you say that it is more normal for PAHs (or dioxin) to suppress estrogen effects and that the effect I allude to (the ability of estrogens to suppress the MFO system) is not the norm (and therefore certainly not accepted as being a 'well-known' mechanism). You sent me back to re-read the Kirby paper and I remain convinced by the data (especially as it was repeated). I also dug out a paper he published in 2004 on EROD measurements in estuaries (field study). He found in that study that where there was a significant difference in EROD activity between males and females (not all estuaries showed a difference) the difference was always males > females (i.e. the field evidence supports the laboratory evidence). He did not distinguish between mature and immature females and this might account for the lack of a difference in some estuaries (i.e. most of the females might have been immature in these estuaries and not making any estradiol).

If I am allowed to speculate, flounders are bottom-dwelling fish that feed on animals living in the mud. Furthermore their habitat is a continental shelf area replete with massive oil and gas reserves. They are more likely to have had to face exposure to PAHs during their time on this planet than, for example, the rainbow trout, which lives in crystal clear mountain streams. This difference in lifestyle might have favoured the evolution of a protective mechanism in the flounder, but not in the rainbow trout (or of course mammals!). Protective of what one might ask? Presumably, if it operates in the mature females only, then it is likely to be the ovaries.

Hi Sandy,

Really interesting question! Regarding flounders (not necessarily conserved in other species), we know that metals can reduce transcription from CYP1A promoter as well, which fits in nicely with Alexander's speculation.

On whether estrogens are really protective versus PAHs, I guess hepatic tumour prevalence over a lifetime might give the answer, and there might be hints in the environmental sampling data (Brett Lyons, Mark Kirby and Grant Stentiford spring to mind, as well as Angela Kohler) but to really do this properly a lifecycle exposure would be needed - tricky in flounder!

One might imagine a slowed metabolism of PAHs leading to lower, repairable levels of DNA damage in the females (or estrogen-exposed) however this contnuing over a longer periood of time.