Effects of persistent organochlorine exposure on the liver transcriptome of the common minke whale (Balaenoptera acutorostrata) from the North Pacific.

Hepatic concentrations of persistent organochlorines (OCs) were determined in the common minke whale (Balaenoptera acutorostrata) from the North Pacific. To investigate the effects of OCs on the transcriptome in the minke whale, the present study constructed a hepatic oligo array of this species where 985 unique oligonucleotides were spotted and further analyzed the relationship between the OC levels and gene expression profiles of liver tissues. The stepwise multiple linear regression analysis identified 32 genes that correlated with hepatic OC levels. The mRNA expression levels of seven cytochrome P450 (CYP) genes, CYP1A1, 1A2, 2C78, 2E1, 3A72, 4A35, and 4V6 showed no clear correlations with the concentration of each OC, suggesting that the accumulated OCs in the liver did not reach levels that could alter CYP expression. Among the genes screened by the custom oligo array analysis, hepatic mRNA expression levels of 16 genes were further measured using quantitative real-time reverse transcription polymerase chain reaction. The mRNA levels of vitamin D-binding protein (DBP) were negatively correlated with non-ortho coplanar polychlorinated biphenyl (PCB) levels. Androgen receptor-associated coregulator 70 (ARA70) expression levels showed a significant positive correlation with concentrations of non-ortho coplanar PCB169. These correlations suggest that coplanar PCB-reduced DBP expression could suppress vitamin D receptor-mediated signaling cascades in peripheral tissues. Alternatively, the suppression of vitamin D receptor signaling cascade could be enhanced through competition with the androgen receptor signaling pathway for ARA70. In addition, a negative correlation between kynureninase and PCB169 levels was also observed, which suggest an enhanced accumulation of an endogenous aryl hydrocarbon receptor agonist, kynurenine in the minke whale population. Further studies are necessary to translate the changes in the transcriptome to toxicological outcomes including the disruption of the nervous and immune systems.

Strain optimization for efficient isobutanol production using Corynebacterium glutamicum under oxygen deprivation.

Microbial production of isobutanol is made difficult by the chemical's high cell toxicity. Corynebacterium glutamicum, inherently one of the more isobutanol-tolerant industrial microorganisms, exhibits unprecedented productivity under oxygen deprivation, potentially allowing for high productivity of such toxic chemicals as isobutanol. Here, we show that development of C. glutamicum strains proficient in isobutanol production depends not only on modulating the activity of 2-keto acid decarboxylase (KDC) and isobutanol dehydrogenase (IBDH) and suppressing by-product formation, but also on optimizing the production process to eschew product inhibition. Isobutanol production under oxygen deprivation reached 343 mM (3.2% v/v) in strain IBU5 expressing kivd (encoding KDC) under the control of ldhA promoter and adhP (encoding IBDH from Escherichia coli MG1655) under the control of gapA promoter. This productivity is double the previously reported best productivity of 1.6% (v/v) and exceeds the 2.5% (v/v) limit beyond which cell growth becomes too severely suppressed. Irrespective, a cumulative 56.5% improvement on yield was possible with the combined effects of disruption of the ppc gene, encoding phosphoenolpyruvate carboxylase (PEPC), use of a NAD⁺-specific mutant acetohydroxyacid isomeroreductase (AHAIR), and overexpression of select glycolytic genes. Using oleyl alcohol to continuously extract the isobutanol from reaction mixture and tripling the cell concentration in the reaction mixture to 60 g dry cell/L stretched the yield to 78.1% and volumetric productivity to 981 mM (9.1% v/v).

Metabolic engineering of 1,2-propanediol pathways in Corynebacterium glutamicum.

We analyzed 1,2-propanediol (1,2-PD) production in metabolically engineered Corynebacterium glutamicum. Wild-type C. glutamicum produced 93 µM 1,2-PD after 132 h incubation under aerobic conditions. No gene encoding the methylglyoxal synthase (MGS) which catalyzes the first step of 1,2-PD synthesis from the glycolytic pathway was detected on the C. glutamicum genome, but several genes annotated as encoding putative aldo-keto reductases (AKRs) were present. AKR functions as a methylglyoxal reductase in the 1,2-PD synthesis pathway. Expressing Escherichia coli mgs gene in C. glutamicum increased 1,2-PD yield 100-fold, suggesting that wild-type C. glutamicum carries the genes downstream of MGS in the 1,2-PD synthesis pathway. Furthermore, simultaneous overexpression of mgs and cgR_2242, one of the genes annotated as AKRs, enhanced 1,2-PD production to 24 mM. This work establishes that 1,2-PD synthesis by C. glutamicum, previously unknown, is possible.

Identification and hepatic expression profiles of cytochrome P450 1-4 isozymes in common minke whales (Balaenoptera acutorostrata).

Full-length cDNA sequences of cytochrome P450 (CYP) 2C78, 2E1, 3A72, 4A35 and 4V6 isozymes were isolated from a hepatic cDNA library of common minke whale (Balaenoptera acutorostrata). The deduced amino acid sequences of minke whale CYP2C78, 2E1, 3A72, 4A35 and 4V6 showed high identities with cattle CYP2C86 (83%), pig CYP2E1 (85%), sheep CYP3A24 (82%), pig CYP4A21 (80%), and human CYP4V2 (76%), respectively. To investigate whether or not these CYP expression levels are altered by contamination of organochlorine contaminants (OCs), mRNA levels of these CYPs in the liver of common minke whale were measured using a quantitative real-time RT-PCR method, and the quantified mRNA levels were employed for the statistical analysis with the residue levels of OCs including PCBs, DDTs (p,p'-DDT, p,p'-DDD and p,p'-DDE), chlordanes (cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor and oxychlordane), HCHs (alpha-, beta- and gamma-isomers) and hexachlorobenzene that have already been reported elsewhere. Spearman's rank correlation analyses showed no significant correlation between CYP expression levels and each OC level in the common minke whale liver, implying that these environmental chemicals have no potential to alter the expression levels of these CYPs or the residue levels encountered in the whale livers may not reach their transcriptional regulation levels. This suggests that the expression of individual CYPs in the whale liver may be at basal level. Relationships among hepatic mRNA expression levels of these CYP2-4 isozymes together with CYP1A1 and CYP1A2 were also examined. Significant positive correlations were detected among mRNA expression levels of individual CYP isozymes in most cases. These associations indicate that the transcriptional regulation of these CYPs examined in this study may be reciprocally related. CYP1A1 levels showed a positive correlation with CYP1A2 levels (r=0.64, p<0.01) indicating that both CYP isozymes were regulated by aryl hydrocarbon receptor activated by endogenous ligands. A strong positive correlation between CYP2C78 and 3A72 (r=0.90, p<0.001) suggests that expression of these CYP isozymes may be under a regulation mechanism of cross-talk in which specific nuclear receptors such as constitutive androstane receptor and pregnane X receptor are involved. The present study indicates that minke whale from the North Pacific may be a model species to investigate the mechanism of basal regulation of these CYPs.

Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in the liver of common minke whales (Balaenoptera acutorostrata).

This study presents full-length cDNA sequences of CYP1A1 and 1A2, in common minke whale (Balaenoptera acutorostrata) from the North Pacific. Both CYP1A1 and CYP1A2 cDNAs had an open reading frame of 516 amino acid residues, and predicted molecular masses were 58.3 kDa and 58.1 kDa, respectively. The deduced full-length amino acid sequence of CYP1A1 revealed higher identities with those of sheep (86%) and pig (87%), and that of CYP1A2 was most closely related to human (82%) and monkey CYP1A2 (82%) among species from which CYP1A2 has been isolated so far. Differences in certain conserved and functional amino acid residues of CYP1A1 and 1A2 between common minke whale and other mammalian species indicate the possibility of their specific metabolic function. Concentrations of organochlorine compounds (OCs) including PCBs and DDTs analyzed in common minke whale liver showed no significant correlation with hepatic mRNA expression levels of CYP1A1 and CYP1A2, indicating no induction of these enzymes by such OCs.

Temporal trends of persistent organochlorine contamination in Russia: a case study of Baikal and Caspian Seal.

To examine temporal trends of organochlorine (OC) contamination in Lake Baikal and the Caspian Sea, concentrations of persistent OCs, such as DDT and its metabolites (DDTs), polychlorinated biphenyls (PCBs), hexachlorocyclohexane (HCHs), chlordane compounds (CHLs), tris(4-chlorophenyl)methane (TCPMe), and tris(4-chlorophenyl)methanol (TCPMOH), in the blubber of female seals were determined. Collections were made in 1992, 1993, 1995 and 1998. DDT concentrations in Baikal and Caspian seals showed a rapid decline during 1992 to 1998, while the concentrations of PCBs declined slowly. Elevated concentrations of HCHs were found in Caspian seals and there was no decline in their concentrations during 1993 to 1998, which could be due to extensive usage of HCHs around Caspian Sea in recent years. Trends of TCPMe and TCPMOH residues in Caspian seals were similar to that of DDTs. The pattern of PCB isomers in both Baikal seals and Caspian seals exhibited little temporal variations. Concentrations of non- ortho coplanar PCBs have declined at a faster rate than those of mono- ortho congeners. Compilation of available data on OC contamination in the North Pacific, Antarctic, Caspian Sea, Lake Baikal, and India suggested that the time trend of residues of contaminants during the 1990s were different among these regions. Residue levels of OC insecticides have declined slowly while PCBs remained at a steady state in the open oceans and the Antarctic. The magnitude of temporal variation in Lake Baikal seemed to be higher than that in the Caspian Sea. Residue concentrations of OCs have increased in Ganges River dolphins from 1989-92 to 1994-96, suggesting that tropical, developing countries are potential emission source of OCs.