Newly Identified CYP2C93 Is a Functional Enzyme in Rhesus Monkey, but Not in Cynomolgus Monkey

Cynomolgus monkey and rhesus monkey are used in drug metabolism studies due to their evolutionary closeness and physiological resemblance to human. In cynomolgus monkey, we previously identified cytochrome P450 (P450 or CYP) 2C76 that does not have a human ortholog and is partly responsible for species differences in drug metabolism between cynomolgus monkey and human. In this study, we report characterization of CYP2C93 cDNA newly identified in cynomolgus monkey and rhesus monkey. The CYP2C93 cDNA contained an open reading frame of 490 amino acids approximately 84–86% identical to human CYP2Cs. CYP2C93 was located in the genomic region, which corresponded to the intergenic region in the human genome, indicating that CYP2C93 does not correspond to any human genes. CYP2C93 mRNA was expressed predominantly in the liver among 10 tissues analyzed. The CYP2C93 proteins heterologously expressed in Escherichia coli metabolized human CYP2C substrates, diclofenac, flurbiprofen, paclitaxel, S-mephenytoin, and tolbutamide. In addition to a normal transcript (SV1), an aberrantly spliced transcript (SV2) lacking exon 2 was identified, which did not give rise to a functional protein due to frameshift and a premature termination codon. Mini gene assay revealed that the genetic variant IVS2-1G>T at the splice site of intron 1, at least partly, accounted for the exon-2 skipping; therefore, this genotype would influence CYP2C93-mediated drug metabolism. SV1 was expressed in 6 of 11 rhesus monkeys and 1 of 8 cynomolgus monkeys, but the SV1 in the cynomolgus monkey was nonfunctional due to a rare null genotype (c.102T>del). These results suggest that CYP2C93 can play roles as a drug-metabolizing enzyme in rhesus monkeys (not in cynomolgus monkeys), although its relative contribution to drug metabolism has yet to be validated

Performance of A-stage process treating combined municipal-industrial wastewater

A biosorption column and a settling tank were operated for 6 months with combined municipal and industrial wastewaters (1 m3/hr) to study the effect of dissolved oxygen (DO) levels and Fe3þ dosage on removal efficiency of dissolved and suspended organics prior to biological treatment. High DO (>0.4 mg/L) were found to be detrimental for soluble chemical oxygen demand (COD) removals and iron dosing (up to 20 ppm) did not improve the overall performance. The system performed significantly better at high loading rate (>20 kg COD.m3.d1) where suspended solids and COD removals were greater than 80% and 60%, respectively. This is a significant improvement compared to conventional primary sedimentation tank and the process is a promising alternative for the pretreatment of industrial wastewater

Development of a new device for artificial insemination in cynomolgus macaques

In cynomolgus macaques, an important animal species for biomedical research, efficient reproduction has been hampered partly due to the difficulties of artificial insemination (AI) using straw tubes developed for humans or farm animals, because cynomolgus macaques have a complex cervical canal structure. In this study, taking into consideration the unique structure of the macaque cervical canal, we developed a novel device for AI, comprised of a syringe and an outer cylinder. At 24 and 48 h after using this device to inject semen into one female, viable sperm were observed in the oviduct where the sperm meets the oocytes. We then attempted AI using this new device on 10 females that were at pre-ovulation, and pregnancy was successful in three animals (30% pregnancy rate). These results show that the newly developed device can be used for AI in cynomolgus macaques

Drug-metabolizing activity of CYP2C93 protein determined using human CYP2C substrates.

<p>CPR, cytochrome P450 reductase. N.D., not determined.</p><p>In each reaction, 5 pmol of the recombinant protein was used with substrate (50 µM diclofenac, 100 µM flurbiprofen, 100 µM paclitaxel, 200 µM <i>S</i>-mephenytoin, or 1 mM tolbutamide) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016923#s2" target="_blank"><i>Materials and Methods</i></a>.</p><p>The recombinant cynomolgus and rhesus monkey CYP2C93 proteins were analyzed along with cynomolgus monkey CYP2C8, CYP2C43, CYP2C75, and CYP2C76. CYP2C93v1 and CYP2C93v2 correspond to SV1 and SV2 transcripts of CYP2C93, respectively.</p

Kinetic analysis for oxidations of typical human CYP2C9 substrates catalyzed by monkey CYP2C93.

<p>Each substrate (0–5000 µM tolbutamide, 0–200 µM diclofenac, and 0–200 µM flurbiprofen) was incubated with recombinant CYP2C93v1 (of rhesus monkey) at 37°C for 15 min in the presence of an NADPH-generating system as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016923#s2" target="_blank"><i>Materials and Methods</i></a>. Kinetic parameters were calculated from a fitted curve by non-linear regression (mean ± SE).</p