Brian G. Lake

The kinetics of metabolism of deltamethrin (DLM) andcis- andtrans-permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol. Apparent intrinsic clearance (CLint) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration. However, when apparent CL(int)values were corrected for nonspecific binding to allow calculation of unbound (i.e., corrected) CL(int)values, the unbound values did not vary greatly with microsomal protein concentration. Unbound CL(int)values for metabolism of 0.05-1 mu M DLM in rat liver microsomes (CYP and CES enzymes) and cytosol (CES enzymes) were not significantly different from rates of DLM metabolism in isolated rat hepatocytes. This study demonstrates that the nonspecific binding of these highly lipophilic compounds needs to be taken into account in order to obtain accurate estimates of rates ofin vitrometabolism of these pyrethroids. While DLM is rapidly metabolisedin vitro, the hepatocyte membrane does not appear to represent a barrier to the absorption and hence subsequent hepatic metabolism of this pyrethroid.

Using a chimeric mouse humanized liver model, we provided evidence that human hepatocytes are refractory to the mitogenic effects of rodent constitutive androstane receptor (CAR) activators. To evaluate the functional reliability of this model, the present study examined mitogenic responses to phenobarbital (PB) in chimeric mice transplanted with rat hepatocytes, because rats are responsive to CAR activators. Treatment with 1000 ppm PB for 7 days significantly increased replicative DNA synthesis (RDS) in rat hepatocytes of the chimeric mice, demonstrating that the transplanted hepatocyte model is functionally reliable for cell proliferation analysis. Treatment of humanized CAR and pregnane X receptor (PXR) mice (hCAR/hPXR mice) with 1000 ppm PB for 7 days significantly increased hepatocyte RDS together with increases in several mitogenic genes. Global gene expression analysis was performed with liver samples from this and from previous studies focusing on PB-induced Wnt/β-catenin signaling and showed that altered genes in hCAR/hPXR mice clustered most closely with liver tumor samples from a diethylnitrosamine/PB initiation/promotion study than with wild-type mice. However, different gene clusters were observed for chimeric mice with human hepatocytes for Wnt/β-catenin signaling when compared with those of hCAR/hPXR mice, wild-type mice, and liver tumor samples. The results of this study demonstrate clear differences in the effects of PB on hepatocyte RDS and global gene expression between human hepatocytes of chimeric mice and hCAR/hPXR mice, suggesting that the chimeric mouse model is relevant to humans for studies on the hepatic effects of rodent CAR activators whereas the hCAR/hPXR mouse is not.

1. A number of chemicals have been shown to produce disruption of the thyroid gland, resulting in reduced thyroid hormone synthesis, by a mechanism involving inhibition of thyroid peroxidase (TPO) activity (EC 1.11.1.8). 2. An assay was developed for rat thyroid gland microsomal TPO activity, employing L-tyrosine as the physiological substrate, with analysis of the formation of the 3-iodo-L-tyrosine (3MIT) metabolite by ultra-performance liquid chromatography-mass spectrometry-mass spectrometry. 3. Formation of 3MIT was linear with respect to both rat thyroid gland microsomal protein concentration and incubation time, whereas only small quantities of 3,5-diodo-L-tyrosine were formed. 4. Studies were performed with nine known TPO inhibitors. The most potent inhibitors were 3-amino-1,2,4-triazole, ethylene thiourea, methimazole and 6-propyl-2-thiouracil which had IC₅₀ values (i.e. concentration to produce a 50% inhibition of enzyme activity) of 0.059, 0.791, 1.07 and 1.96 μM, respectively, whereas the least potent inhibitor was sodium perchlorate which had an IC₅₀ value of 13,800 µM. 5. For five inhibitors, where literature data were available, the observed IC₅₀ values obtained in this study employing rat thyroid gland microsomes and L-tyrosine as substrate were similar to those previously reported using the spectrophotometric guaiacol oxidation assay.

(2018). Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes. Xenobiotica. Ahead of Print.

The metabolism of deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in liver microsomes, liver cytosol and plasma from male Sprague–Dawley rats aged 15, 21 and 90 days...

A large number of nongenotoxic chemicals have been shown to increase the incidence of liver tumours in rats and/or mice by a mode of action (MOA) involving activation of the constitutive androstane receptor (CAR). Studies with the model CAR activator phenobarbital (PB) and its sodium salt (sodium phenobarbital; NaPB) have demonstrated that the key and associative events for rat and mouse liver tumour formation include CAR activation, increased hepatocyte replicative DNA synthesis (RDS), induction of cytochrome P450 CYP2B subfamily enzymes, liver hypertrophy, increased altered hepatic foci and hepatocellular adenomas/carcinomas. The key species difference between the rat and mouse compared to humans, is that human hepatocytes are refractory to the mitogenic effects of PB/NaPB and other CAR activators. While PB/NaPB and other CAR activators stimulate RDS in rat and mouse hepatocytes in both in vitro and in vivo studies, such compounds do not stimulate RDS in cultured human hepatocytes and in in vivo studies performed in chimeric mice with humanised livers. In terms of species differences in RDS, unlike the rat and mouse, humans are similar to other species such as the Syrian hamster and guinea pig in being nonresponsive to the mitogenic effects of CAR activators. Overall, the MOA for rat and mouse liver tumour formation by PB/NaPB and other CAR activators is considered qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies, which demonstrate that chronic treatment with PB does not increase the incidence of liver cancer in humans.

1. The aim was to employ real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) technology (TaqMan ®) to examine the induction of some selected cytochrome P450 (CYP) forms in precision-cut rat liver slices. 2. Taqman ® primers and probe sets were developed for rat CYP1A1, CYP1A2, CYP2B1 and CYP4A1 forms. 3. Rat liver slices were cultured in control medium or medium containing either 10 µg ml -1 Aroclor 1254 (ARO), 500µM sodium phenobarbitone (NaPB) or 50µM Wy-14,643 (WY) for 3, 6 and 24 h. 4. Compared with control liver slices, treatment with ARO for 3 and 6 h produced 24- and 184-fold increases, respectively, in CYP1A1 mRNA levels, and after 24h produced an 85-fold increase in CYP1A2 mRNA levels. Levels of CYP1A1 and CYP1A2 mRNA were not markedly affected by NaPB and WY. 5. Treatment with ARO and PB for 24 h produced 10.6- and 23.8-fold increases, respectively, in CYP2B1 mRNA. Levels of CYP2B1 mRNA were not markedly affected by WY. 6. Treatment with WY, but not ARO and NaPB, for 24h produced a 20.4-fold increase in levels of CYP4A1 mRNA. 7. These results demonstrate that cultured liver slices may be used to evaluate the effect of xenobiotics on CYP form mRNA levels.

Olanzapine metabolism was investigated using incubation of olanzapine with human liver slices. The intent of the investigation was to identify olanzapine metabolites and determine if the human liver slice incubations could potentially produce quantities of the olanzapine glucuronides for future studies. Along with known Phase 1 olanzapine metabolites, N‐desmethyl‐, 2‐hydroxymethyl‐, and 4′‐N‐oxide‐, a new hydroxylated species was detected. Detection of Phase 2 metabolites included known N‐10‐glucuronides, a quaternary glucuronide and a novel glucuronide conjugate. This investigation showed the feasibility of using human liver slices to produce sufficient quantities of olanzapine glucuronides for further studies. © 1998 John Wiley & Sons, Ltd.