Inhibitory and inductive effects of 4- or 5-methyl-2-mercaptobenzimidazole, thyrotoxic and hepatotoxic rubber antioxidants, on several forms of cytochrome P450 in primary cultured rat and human hepatocytes

Highlights • 4-MeMBI, 5-MeMBI and MBI inhibit CYP3A2 activity in cultured rat hepatocytes.• 4-MeMBI and 5-MeMBI, not MBI, induce CYP1A1/2 activity in cultured rat hepatocytes.• Effects of the chemicals on CYP3A2 suggest metabolic drug-drug interaction potential.• 4-MeMBI and 5-MeMBI induce CYPs 3A4 and 1A1/2 activity in cultured human hepatocytes.• Primary cultured hepatocytes are useful in CYP experiments of benzimidazole compounds.

The above findings suggest that the effects of 4-MeMBI and 5-MeMBI on hepatic drug-metabolizing activity is different from typical thioureylene compounds such as methimazole, which strongly inhibits the activity of several human haptic microsomal drug-metabolizing enzymes including CYP3A4 [10]. This is very important because CYP3A4, the human homologue of rat CYP3A2, is a major CYP in the liver [11] and is therefore involved in metabolic drug-drug interactions [12]. However, the effects of 4-MeMBI and 5-MeMBI on CYP3A2/4 were not fully examined; no protein expression changes in CYP3A2 were observed by semi-quantitative western blot analysis in a rat in vivo experiment [8]. In addition, possible inducibility of CYP3A2/4 by 4-MeMBI and 5-MeMBI should be taken into account when their effects are examined, as suggested from the inducibility of CYP3A4 by benzimidazole compounds [13].
These metabolic and toxicologic studies of methyl-MBIs and MBI are important because metabolic drug-drug interaction can be further complicated by other modifier of CYP3A4 activity including easily available supplements such as Terminalia arjuna products [14]. In addition, benzimidazole compounds has common toxicological effects, such as oxidative stress and apoptosis [15], which can cause mechanistic  drug-drug interaction.
In the present study, we examined the effects of 4-MeMBI, 5-MeMBI and MBI on the activity of CYPs 3A2 and 1A1/2 in primary cultured rat hepatocytes, which enable experiments under several exposure conditions with fewer animals than in in vivo experiments. Primary rat hepatocytes were cultured for 48 or 96 h in the presence of 4-MeMBI, 5-MeMBI or MBI and the activity of CYPs 3A2 and 1A1/ 2 was determined by measuring activity of testosterone 6β-hydroxylation (T6βH) [16] and 7-ethoxyresorufin O-deethylation (EROD) [17], respectively. CYP1A1/2 was selected for validation of the primary cultured rat hepatocytes as an in vitro model of rat liver because it is strongly induced by 4-MeMBI and 5-MeMBI, but not by MBI, in vivo [8]. With a limited number of donors, inducibility of 4-MeMBI, 5-MeMBI and MBI in primary cultured human hepatocytes was also examined for comparative metabolism studies.

Chemicals
The structures of the 4-MeMBI, 5-MeMBI and MBI are shown in

Culture of rat hepatocytes
Rat hepatocytes were prepared by the two-step collagenase perfusion method [19] from male Wistar rats (6-weeks old) purchased from Japan SLC Inc., (Shizuoka, Japan). The hepatocytes were seeded into 24-well culture plates coated with collagen (BioCoat Collagen I 24-well plate, Corning Inc., Corning, NY, USA) at a cell density of 2 × 10 5 cells/cm 2 in the Lanford medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and allowed to attach to the culture plate during pre-incubation for 3 h at 37 • C with 5% CO 2 . After the pre-incubation, the culture medium was replaced with 0.25 mL/well of the Lanford medium containing designated concentration of chemicals. The hepatocytes were cultured in the presence of chemicals for 48 or 96 h, during which the culture medium was replaced every 24 h. All animal experiments were carried out according to the guidelines for animal use of the National Institute of Health Sciences.

Culture of human hepatocytes
Freshly isolated human hepatocytes from two donors, Sau01 (Caucasian male, 17-years old, smoking 1 pack per day for 1 year) and Sau06 (Caucasian female, 17-years old, no smoking history), were purchased (Gentest, Corning) as attached cells to 24-well culture plates (BioCoat Collagen I 24-well plate, Corning) at a cell density of 2 × 10 5 cells/cm 2 . The hepatocytes were pre-cultured in the Lanford medium, which was replaced every 48 h, at 37 • C with 5% CO 2 for 1 week prior to the experiments. After the pre-culture, the culture medium was replaced with 0.25 mL/well of the Lanford medium containing designated concentration of chemicals. The hepatocytes were cultured in the presence of chemicals for 48 or 96 h, during which the culture medium was exchanged every 24 h. Effects of MBI on human hepatocytes were not examined because of the scarcity of available cells. The human hepatocytes used in the present study were purchased from the Gentest, and therefore, the study was in accordance with The Declaration of Helsinki developed by the World Medical Association. For the same reason, the code of ethics of the National Institute of Health Sciences is not applicable.

Statistical analysis
Statistical significance of the differences between the experimental groups was examined by the one-way analysis of variance with the Dunnett's multiple comparison test at probability levels of 5% and 1%.

Effects on CYP3A2 activity in primary cultured rat hepatocytes
4-MeMBI, 5-MeMBI and MBI (≥12.5 μM) reduced CYP3A2 activity by more than 50 % in a concentration-dependent manner ( Fig. 2A-C). CYP3A2 activity measured at 96 h, however, was generally higher than that at 48 h over the concentration range of 4-MeMBI, 5-MeMBI and MBI used, suggesting an exposure time-dependent weak inducibility of CYP3A2 ( Fig. 2A-C). Dexamethasone, a known inducer of CYP3A2, increased CYP3A2 activity at 96 h but not at 48 h, supporting the inducibility of CYP3A2 by 4-MeMBI, 5-MeMBI and MBI in an exposure time-dependent manner (Fig. 2D).

Discussion
The present results indicate that 4-MeMBI, 5-MeMBI and MBI inhibit CYP3A2 activity, which can cause metabolic drug-drug interactions. It is considered that the inhibitory effects on CYP3A2 is due to the thioureylene structure, because 4-MeMBI, 5-MeMBI and MBI share this structure, and methimazole, a typical thioureylene compound, inhibits the activity of CYP3A4, the human homolog of rat CYP3A2, in human microsomes [10].
On the other hand, there may be time-dependent weak inductive effects due to the MBI structure as a common profile among 4-MeMBI, 5-MeMBI and MBI, since omeprazole and lansoprazole, antiulcer drugs containing the MBI structure, cause the dual opposite effects on CYP3A4 activity, i.e., inhibition in human microsomes and induction in primary cultured human hepatocytes [13,21].
The above results also indicate the usability of primary cultured rat hepatocytes to examine the effects of 4-MeMBI, 5-MeMBI and MBI on CYP3A2 activity, which might not be observed in in vivo studies. This is because these effects were concentration-and time-dependent, and therefore must have required large number of experimental animals if examined in in vivo studies. It is suggested that CYP3A2 protein reduced only by MBI in the in vivo experiment is due to other factors such as their metabolic profiles; e.g., detoxifying desulfurization was reduced for MBI, but was enhanced for 4-MeMBI and 5-MeMBI, by repeated administration in rats [7,8].
As for CYP1A1/2, 4-MeMBI and 5-MeMBI, but not MBI, induced CYP1A1/2 in the present study; the former was more potent than the latter. These results are consistent with the in vivo results where repeated oral administration of either 4-MeMBI or 5-MeMBI induced CYP1A1/2 as determined by EROD and western blot analysis in rat liver microsomes [8]. This consistency validates primary cultured rat hepatocytes as an experimental model for the investigation of 4-MeMBI, 5-MeMBI and MBI.
It is considered that, if any, the different effects on CYPs among 4-MeMBI, 5-MeMBI and MBI both in rat and human hepatocytes were attributable to the presence and position of the methyl group, their sole structural difference. However, there seems no clear relationship between the methylated benzene ring of these chemicals and their inhibitory effects on CYP3A2/4 activity. On the other hand, the different effects of these chemicals confirmed the importance of the substituted benzene ring in the potent inducibility of CYP1A1/2 by benzimidazole compounds in cultured hepatic cells. In cultured rat hepatoma cells, 5methoxy-MBI induced CYP1A1 protein more potently than MBI [22]. Omeprazole, which has a methoxy group on the benzene ring, induced CYP1A1 more potently than lansoprazole, which has no substituted benzene ring, in cultured hepatic cells [13,22]. Since the inducibility of CYP1A1 by omeprazole depends on the activation of aryl hydrocarbon receptor (AhR), it is supposed that methylated benzene ring strengthens the potency of methyl-MBIs to activate AhR in an order of 4-methyl > 5-methyl in rats, but 5-methyl > 4-methyl in humans.
When rat and human hepatocytes are compared, there seem to be differences in the effects of 4-MeMBI, 5-MeMBI and MBI on CYP activity. This is because inhibitory effects on CYP3A2 of 4-MeMBI and 5-MeMBI observed in rat hepatocytes, were not observed in human hepatocytes. On the other hand, the inducibility of CYP1A1/2 was more potent by 4-MeMBI than by 5-MeMBI in rat hepatocytes, but 5-MeMBI was more potent in human hepatocytes. These differences may be related to known species differences in the inducibility of CYPs [23].
The present results provide some insights into induction mechanisms of CYPs 3A4 and 1A1/2 in human hepatocytes although inconclusive because of the small number of donors. For example, the higher induction of CYP3A4 by 4-MeMBI and 5-MeMBI in the non-responder to rifampicin (Sau06) suggests that the inducibility is pregnane X receptor (PXR)-independent because rifampicin is a PXR-dependent inducer [24]. Similarly, the higher induction of CYP1A1/2 by 4-MeMBI and 5-MeMBI in the low responder to omeprazole (Sau06) suggests that the inducibility is different from that by omeprazole, an inducer known to involve AhR [25].
In terms of comparative metabolism, the induction of CYP1A1/2 activity by 4-MeMBI and 5-MeMBI in rat versus human hepatocytes suggests enhanced detoxification of these chemicals in humans. This is because repeated administration of these chemicals to rats increases hepatic CYP1A1/2 activity and urinary excretion of less toxic desulfurated metabolites in vivo [7,8], and because rat hepatic CYP1A metabolizes these chemicals in vitro [9]. It was thus considered that 4-MeMBI and 5-MeMBI could be detoxified by CYP1A1/2 induced in the human liver.
It should be noted, however, that the above findings based on human hepatocytes in the present experiment are inconclusive. This is because the number of human donors is as small as two and because possible loss of responsiveness of CYP3A4 to rifampicin as observed in one donor, although the non-responsiveness could be due to great individual variability [26].
In conclusion, 4-MeMBI, 5-MeMBI and MBI cause inhibition of CYP3A2 in primary cultured rat hepatocytes, suggesting their potential for metabolic drug-drug interactions. Primary cultured rat and human hepatocytes are considered to be useful for the evaluation of effects of the benzimidazole compounds on their inducibility and inhibitory activities of cytochrome P450 forms.

Funding
This work was partly supported by a Health and Labor Science Research Grant from the Ministry of Health, Labor and Welfare in Japan.

Declaration of Competing Interest
The authors report no declarations of interest.