Application of new approach methodologies in molecular toxicology
Authors:   , Santos Capinha Liliana Monica 
Published: January 2024

Traditional safety assessments often rely on animal models, which may not reflect human responses to chemical exposures and raise ethical concerns. Despite the increasing efforts to develop new approach methodologies (NAMs), their integration into regulatory decision-making processes is still challenging. The European Union has played a key role in the advancement of NAMs through funding projects like the EU-ToxRisk project. This PhD thesis, funded by EU-ToxRisk, focuses on the multi-organ metabolism of chemicals like trichloroethylene (TCE), a halogenated alkene which has been extensively associated with toxicity and carcinogenicity. TCE’s nephrotoxic effects result from multi-organ metabolism and bioactivation via the mercapturic acid pathway.
Based on its chemical structure, substitution of chlorine-atoms of TCE by glutathione (GSH) theoretically can lead to the formation of three regioisomeric GSH-conjugates: S-(1,2-trans-dichlorovinyl)-glutathione (1,2-trans-DCVG), S-(1,2-cis-dichlorovinyl)-glutathione (1,2-cis-DCVG) and S-(2,2-dichlorovinyl)-glutathione (2,2-DCVG). However, at the time of the start of this PhD project, there was only experimental evidence for two regioisomers, namely 1,2-trans-DCVG and 2,2-DCVG.
In chapter 2, a novel LC-MS method was developed to separate and quantify DCVG regioisomers and their corresponding cysteine S-conjugate (DCVCs) and N-acetyl-L-cysteine-S-conjugates (DCV-NACs). This method revealed significant differences in the formation profile of these regioisomers when comparing incubations in rat and human liver fractions. Novel methods were also developed for the synthesis and purification of all three regioisomers of DCVG, DCVC, and DCV-NAC. Using a β-lyase-mimetic model and 4-(p-nitrobenzyl)pyridine (NBP) as a model nucleophile, we demonstrated that only 1,2-trans-DCVC and 1,2-cis-DCVC formed NBP-reactive products, suggesting their higher reactivity.
The purified regioisomers of DCVG and DCVC were used to investigate their cellular effects in different cellular models. In chapter 3, six different human in vitro systems representing target organs of TCE were used to compare altered transcriptional responses upon exposure to the major conjugates formed by human liver fractions, 1,2-trans-DCVG/C and 2,2-DCVG/C (chapter 2). Viability studies showed that 1,2-DCVC affected the viability of all six cell models in a concentration-dependent manner, while 1,2-DCVG only affected the kidney model (RPTEC/TERT1). The kidney and liver models showed the strongest transcriptomic responses to both 1,2-DCVG and 1,2-DCVC. Transcripts associated with Nrf2-mediated oxidative stress response and ATF4-mediated unfolded protein response (UPR) were upregulated in all cell types at high concentrations of 1,2-DCVC exposure. In chapter 4, the renal cellular effects of 1,2-cis-DCVC and 1,2-cis-DCVG were investigated, showing significant differences in their effects on cell viability and mitochondrial respiration compared to 1,2-trans-DCVG/C and 2,2-DCVG/C isomers. Despite similar reactivities of the β-lyase products of 1,2-trans-DCVC and 1,2-cis-DCVC, 1,2-cis-DCVC did not affect cell viability of RPTEC/TERT1 cells at any of the concentrations tested. The rates of metabolism of the three DCVG regioisomers by RPTEC/TERT1 cells were investigated. All three DCVGs were degraded rapidly at comparable rates, with simultaneous formation of their corresponding cysteinylglycinyl-S-conjugates (DCV-CysGly). Three metabolites corresponding to N-γ-glutamyl-S-(dichlorovinyl)-L-cysteine (DCV-CysGlu) conjugates were identified, marking the first time this pathway is reported in TCE metabolism.
In chapter 5, cellular responses were determined after repeated daily exposure for 7 days to non-toxic concentrations of TCE-conjugates. Both 1,2-trans-DCVC and 1,2-trans-DCVG started to cause a decrease in cell number after 5 days of exposure, with 1,2-trans-DCVC being slightly more toxic. 2,2-DCVG, the major DCVG-regioisomer formed by human liver fractions (chapter 2), only produced minor Nrf2-responses when exposed for 24 h at high concentration (>250 µM) to RPTEC/TERT1-cells. This confirms that this regioisomer seems to be of low toxicological concern for humans.
In conclusion, by integrating kinetic and dynamic techniques for the evaluation of compounds that go through the GSH conjugation pathway, the work done in this PhD thesis offers confidence in the successful application of NAMs to support chemical risk assessment.