Future considerations

This project had a number of limitations that may have impacted the in vivo relevance of the results obtained and which should be considered in future studies. The doses used in this study were chosen after having been demonstrated to induce cytotoxicity and were therefore not necessarily pharmacologically relevant.26, 117 Whilst this allowed for the assessment of which biochemical pathways may have been affected in hepatocytes during GTE-induced hepatotoxicity, it may not be completely demonstrative of what occurs in individuals who develop liver damage in response to this HCAM. The length of treatment must also be taken into account when considering dosage; due to the limited timeframe, this study focused on acute, high doses in order to elicit a timely toxic response. Future studies should use

pharmacological doses and chronic exposure periods in order to better represent the conditions under which cases of GTE-induced liver damage have manifested.3, 4

Bioavailability must also be taken into consideration, given the low oral bioavailability previously demonstrated in GTE components such as catechins, particularly when consumed in a fed state.33 _{Additionally, microbial metabolism of} catechins results in a variety of conjugations and thus may impact their overall bioavailability.61 _{It is therefore likely that the GTE components and metabolites to} which cells were exposed in this study were not a complete representation of what they may come into contact with in vivo.

Whilst analysing hepatocyte metabolism in vitro is achievable, it is not possible to completely assess the pharmacokinetics of GTE using this model. Understanding the absorption, distribution and excretion of GTE constituents and metabolites in addition to their metabolism will be paramount to understanding and predicting the risk of toxicity induced by GTE, or other herbal medicines, in certain individuals. In addition to the pharmacology of GTE and its constituents, there are other factors which may contribute to the toxicity observed in certain individuals which could not be sufficiently examined in the timeframe of this study. For example, it has been demonstrated that the contamination and adulteration of herbal supplements is not only possible, but rife among these products.12, 144 _{Given that in many instances of} liver damage the GTE product consumed has not been available for testing, it is difficult to rule out contamination or adulteration of these products leading to the liver damage reported. Given that this study did not characterise the composition of

the GTE product prior to cell exposures, contamination or adulteration cannot be ruled out as a possible contributor to the cytotoxicity observed.

Genetic variation may also play a role in determining which individuals may be more predisposed to developing a liver injury when consuming GTE.34, 35, 86 _{For example,} polymorphisms resulting in altered function or abundance of the enzymes responsible for the biotransformation of catechins may increase the likelihood of hepatotoxicity due to the potentially toxic accumulation of these compounds or the increased production of toxic metabolites.34 _{Additionally, changes in the proteins} involved in Phase III detoxification could limit the clearance of catechins and metabolites from the liver, causing them to build up, potentially resulting in hepatotoxicity.24, 25 _{Future studies should examine whether any common genetic} abnormalities are able to be identified amongst individuals who develop liver injury following supplementation with this HCAM.

Co-medication with other herbal supplements or drugs could result in herb-herb or herb-drug interactions, which may contribute to certain individuals being more susceptible to developing GTE-induced liver damage.16, 17 _{In fact, a number of cases} of liver damage in which GTE was identified as the most likely culprit have been in individuals using a supplement which contained GTE as one of many ingredients.4 _It is possible that GTE could interact with other components present in these products to cause liver injury, and may not be as damaging when consumed in isolation at a safe dosage. Further research should be conducted in order to determine the composition of these products and whether the hepatotoxicity of these substances can really be narrowed down to a single component.

If this study were to be repeated, it may also be beneficial to use a different hepatocyte cell line, such as HepaRG. It is well established that HepG2 cell responses are not completely comparable to in vivo hepatocyte responses; for example, the diminished function of the CYP450 enzymes is a particular setback when assessing toxicity with this cell line.5 _{HepaRG cells are an immortalised cell line which have} retained metabolic function, therefore these may be a better choice in future.5 Additionally, using 3D cultures that include non-parenchymal liver cells may produce results more comparable to in vivo responses thanthe traditional 2D culture used in this study, given that they allow more complex cell-cell interactions.145 _{Of course, the} gold standard would be to obtain primary human hepatocyte cultures, however this is unlikely to be an option until further evidence is gathered.5, 145

A major challenge with GC-MS analysis is the formation of multiple derivatisation products. This makes it challenging to gain a complete understanding of a given metabolic profile due to the production of multiple results for the same compound. In this study, the method of overcoming this without further analysis was to select the result that appeared to contribute most to the variation between samples, and disregard the others. This is flawed and thus it would be more suitable to conduct further analysis using other forms of metabolomic analysis such as LC-MS and NMR spectroscopy, the complementarity of which was discussed in a 2018 systematic review.5