Letter to the editor regarding the review article by Yamada et al. (2021) titled “Critical evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR)”

We read with considerable interest the comprehensive review article by Yamada et al. (2021) on a topic that is a matter of scientific debate for quite some years now: the human relevance of the mode of action (MoA) for liver tumor formation by chemicals that are activators of the constitutive androstane receptor (CAR). Following a critical analysis of currently available data concerning this topic the authors conclude that this MoA is qualitatively not plausible for humans. Their conclusion is based on a pivotal species difference in hepatocellular proliferation (a key event in CAR-mediated rodent liver tumor formation), supported by negative epidemiology data for the archetypical CAR activator phenobarbital (PB). According to the authors, CAR activators are mitogenic agents in rodent but not in human hepatocytes, as evidenced by a lack of replicative DNA synthesis (RDS) stimulation in in vitro studies using cultured human hepatocytes and in in vivo studies with chimeric mice bearing human hepatocytes. We believe that the advocated use of these two experimental systems in demonstrating species (dis)concordance with respect to the proliferation key event in the CAR MoA merits a response. In our view, a human relevance analysis should be conducted in a transparent and systematic manner, taking into account both toxicodynamic and -kinetic data. Furthermore, there is currently insufficient insight into the predictive value of the two experimental systems with human hepatocytes and into their robustness as to proliferation induced by hepatocarcinogenic chemicals.

Predictive value

According to Yamada et al. (2021), human CAR supports the hypertrophic, but not the hyperplastic response to PB and other non-genotoxic CAR activators. Hence, they conclude that CAR activators will not be carcinogenic in humans, since a proliferative effect is a necessary key event for liver tumor formation. As support for their conclusion the authors point to several human epidemiology studies showing no increased risk of liver tumors (or tumors in other organs). However, these epidemiology data mostly concern PB, hardly any other CAR activators, and in contrast to PB no data on proliferation in the two experimental systems are available for these few other CAR activators. This renders the evidence base in terms of epidemiological data problematically small, with a view to both the negative and positive predictive value of the two experimental systems used for empirical observations. For the applied negative control PB, which based on current epidemiological data seems not to be a liver carcinogen in humans treated with this drug, the authors have illustrated that PB consistently produces negative results for RDS induction in human hepatocytes. However, information on the positive predictive value of the two experimental systems with human hepatocytes for known human liver carcinogens is not presented. To the best of our knowledge, such information has also not been reported elsewhere in literature. The authors do remark in their review article that “no chemicals have been reported to induce RDS in human hepatocytes”, but details on, for example, the number and type of chemicals tested are lacking. Consequently, the question to what extent the two experimental systems with human hepatocytes are fit-for-purpose for a species concordance analysis of the proliferation key event in the MoA under debate remains unanswered.

One can also question the relevance of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) as positive controls in the systems with human hepatocytes, in absence of a suitable chemical. Yamada and coworkers justify the use of these growth factors as positive control with the argument that EGF and HGF are known hepatocyte mitogens and thus can serve as markers of the functional viability and responsiveness of human hepatocytes to mitogenic stimuli. Whereas this may be true, it is unclear how similar these growth factors and liver carcinogens are in their MoA as to for example receptor activation and triggering subsequent cell proliferation. In other words, how predictive is RDS induction in human hepatocytes by growth factors for RDS induction by (human) liver carcinogens.

Proliferation in cultured primary human hepatocytes and in chimeric mice with human hepatocytes

The review article by Yamada et al. (2021) mentions that cultured human hepatocytes have been extensively used by many laboratories and are considered the “gold standard” in vitro system for many applications. Whereas primary human hepatocytes may indeed be a valuable in vitro system for toxicity testing and investigations relating to hepatic metabolism, it is unclear whether they are suited for the evaluation of hepatocellular proliferation. As shown by among others Godoy et al. (2013), the process of isolation, preservation and culturing of primary human hepatocytes is complex, their quality is highly donor-dependent, and their functionality can be compromised. Ongoing discussions with regard to the use of human hepatocytes for toxicity testing also relate to considerations such as the minimum number of donors to be used, the age range of the donors and whether always cells from both sexes should be used in order to have a credible test model.

In addition to in vitro studies using primary human hepatocytes, Yamada et al. (2021) consider the chimeric mouse model with donor hepatocytes from humans and rats a reliable and scientific credible in vivo approach to examine the effects of chemicals. Their conclusion appears to be based on three studies, in which treatment of chimeric mice with human hepatocytes with PB and two other CAR activators did not result in a stimulation of RDS in human hepatocytes, whereas treatment with EGF did (Yamada et al. 2014; Okuda et al. 2017), as did treatment of chimeric mice with rat hepatocytes with PB and EGF (Yamada et al. 2020). We consider the authors’ claim as not fully justified at this point in time, given that the abovementioned issues such as quality and functionality of human hepatocytes, number of donors used and the use of EGF as positive control also apply to the experimental model with chimeric mice. Further, the Okuda et al. 2017 study indicates that given the relatively low number of animals used in experiments with chimeric mice, as well as the considerable inter-animal variability in response, potential increases in RDS induction may not reach statistical significance and hence may go unnoticed. In combination with the lower proliferation level in human hepatocytes as compared to rat hepatocytes, also evident from the studies by Yamada et al. (2014, 2020), this may lead to false negative results for RDS induction in human hepatocytes and erroneously raise the impression of a qualitative difference between humans and rats.

Conclusion

In order to reach the conclusion that a MoA for adverse health effects is qualitatively not plausible for humans and thus can be disregarded for human health risk assessment, it is crucial to demonstrate substantial species differences through a systematic weight of evidence assessment of that particular MoA. Based on the arguments presented above, we are of the opinion that it is too premature to support the conclusion by Yamada et al. (2021) that such a species difference is evident for CAR-mediated liver tumor formation. In our view, the authors’ claim that the two test systems discussed are valid systems to permit the evaluation of species differences in the effects of CAR activators on hepatocyte proliferation, is not sufficiently substantiated yet. Not only is it questionable how robust these systems are for investigating proliferative effects, but also the evidence base to support their predictive value in relation to RDS induction is (far) too small. Hence, a further evaluation and validation of the two test systems is warranted before the results can be used in a weight of evidence assessment on the human relevance of CAR-mediated liver tumor formation.

Declaration of Interest

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was funded by the Dutch Ministry of Infrastructure and Water Management (I&W). The funding body had no involvement in the design and writing of the letter to editor, and in the decision to submit the letter for publication.