Effects of Subnormothermic Regulated Hepatic Reperfusion on Mitochondrial and Transcriptomic Profiles in a Porcine Model.

OBJECTIVE: We sought to investigate the biological effects of pre-reperfusion treatments of the liver after warm and cold ischemic injuries in a porcine donation after circulatory death model. SUMMARY OF BACKGROUND DATA: Donation after circulatory death represents a severe form of liver ischemia and reperfusion injury that has a profound impact on graft function after liver transplantation. METHODS: Twenty donor pig livers underwent 60 minutes of in situ warm ischemia after circulatory arrest and 120 minutes of cold static preservation prior to simulated transplantation using an ex vivo perfusion machine. Four reperfusion treatments were compared: Control-Normothermic (N), Control- Subnormothermic (S), regulated hepatic reperfusion (RHR)-N, and RHR-S (n = 5 each). The biochemical, metabolic, and transcriptomic profiles, as well as mitochondrial function were analyzed. RESULTS: Compared to the other groups, RHR-S treated group showed significantly lower post-reperfusion aspartate aminotransferase levels in the reperfusion effluent and histologic findings of hepatocyte viability and lesser degree of congestion and necrosis. RHR-S resulted in a significantly higher mitochondrial respiratory control index and calcium retention capacity. Transcriptomic profile analysis showed that treatment with RHR-S activated cell survival and viability, cellular homeostasis as well as other biological functions involved in tissue repair such as cytoskeleton or cytoplasm organization, cell migration, transcription, and microtubule dynamics. Furthermore, RHR-S inhibited organismal death, morbidity and mortality, necrosis, and apoptosis. CONCLUSION: Subnormothermic RHR mitigates IRI and preserves hepatic mitochondrial function after warm and cold hepatic ischemia. This organ resuscitative therapy may also trigger the activation of protective genes against IRI. Sub- normothermic RHR has potential applicability to clinical liver transplantation.

The Microbiota of Freshwater Fish and Freshwater Niches Contain Omega-3 Fatty Acid-Producing Shewanella Species.

Approximately 30 years ago, it was discovered that free-living bacteria isolated from cold ocean depths could produce polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) (20:5n-3) or docosahexaenoic acid (DHA) (22:6n-3), two PUFA essential for human health. Numerous laboratories have also discovered that EPA- and/or DHA-producing bacteria, many of them members of the Shewanella genus, could be isolated from the intestinal tracts of omega-3 fatty acid-rich marine fish. If bacteria contribute omega-3 fatty acids to the host fish in general or if they assist some bacterial species in adaptation to cold, then cold freshwater fish or habitats should also harbor these producers. Thus, we undertook a study to see if these niches also contained omega-3 fatty acid producers. We were successful in isolating and characterizing unique EPA-producing strains of Shewanella from three strictly freshwater native fish species, i.e., lake whitefish (Coregonus clupeaformis), lean lake trout (Salvelinus namaycush), and walleye (Sander vitreus), and from two other freshwater nonnative fish, i.e., coho salmon (Oncorhynchus kisutch) and seeforellen brown trout (Salmo trutta). We were also able to isolate four unique free-living strains of EPA-producing Shewanella from freshwater habitats. Phylogenetic and phenotypic analyses suggest that one producer is clearly a member of the Shewanella morhuae species and another is sister to members of the marine PUFA-producing Shewanella baltica species. However, the remaining isolates have more ambiguous relationships, sharing a common ancestor with non-PUFA-producing Shewanella putrefaciens isolates rather than marine S. baltica isolates despite having a phenotype more consistent with S. baltica strains.

Gene expression and pathologic alterations in juvenile rainbow trout due to chronic dietary TCDD exposure.

The goal of this project was to use functional genomic methods to identify molecular biomarkers as indicators of the impact of TCDD exposure in rainbow trout. Specifically, we investigated the effects of chronic dietary TCDD exposure on whole juvenile rainbow trout global gene expression associated with histopathological analysis. Juvenile rainbow trout were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb (ngTCDD/g food), and fish were sampled from each group at 7, 14, 28 and 42 days after initiation of feeding. 100 ppb TCDD caused 100% mortality at 39 days. Fish fed with 100 ppb TCDD food had TCDD accumulation of 47.37 ppb (ngTCDD/g fish) in whole fish at 28 days. Histological analysis from TCDD-treated trout sampled from 28 and 42 days revealed that obvious lesions were found in skin, oropharynx, liver, gas bladder, intestine, pancreas, nose and kidney. In addition, TCDD caused anemia in peripheral blood, decreases in abdominal fat, increases of remodeling of fin rays, edema in pericardium and retrobulbar hemorrhage in the 100 ppb TCDD-treated rainbow trout compared to the control group at 28 days. Dose- and time-dependent global gene expression analyses were performed using the cGRASP 16,000 (16K) cDNA microarray. TCDD-responsive whole body transcripts identified in the microarray experiments have putative functions involved in various biological processes including growth, cell proliferation, metabolic process, and immune system processes. Nine microarray-identified genes were selected for QPCR validation. CYP1A3 and CYP1A1 were common up-regulated genes and HBB1 was a common down-regulated gene among each group based on microarray data, and their QPCR validations are consistent with microarray data for the 10 and 100 ppb TCDD treatment groups after 28 days exposure (p<0.05). In addition, in the 100 ppb group at 28 days, expression of complement component C3-1 and trypsin-1 precursor have a more than 10-fold induction from the microarray experiments, and their QPCR validations are consistent and showed significant induction in the 100 ppb group at 28 days (p<0.05). Overall, lesion in nasal epithelium is a novel and significant result in this study, and TCDD-responsive rainbow trout transcripts identified in the present study may lead to the development of new molecular biomarkers for assessing the potential impacts of environmental TCDD on rainbow trout populations.

Fish ingestion and congener specific polychlorinated biphenyl and p,p'-dichlorodiphenyldichloroethylene serum concentrations in a great lakes cohort of pregnant African American women.

A cohort of low income, city dwelling, pregnant African American Women (delivered from 1994-1999) was assembled to identify factors related to organochlorine exposure through consumption of Great Lakes resources. The cohort is known as the Great Lakes Cohort of Pregnant African American Women (GLCPAAW). Pregnant women from metropolitan Chicago, IL area clinics were administered a questionnaire on diet, demographics, and health history. Weight, height, and serum lipids were measured at delivery along with serum organochlorines such as PCBs and DDE. Congener specific concentrations of PCBs and p,p'-DDE found in the maternal serum are reported. Dominant PCB congeners found in the serum of the pregnant women at delivery included PCB 101, 118, 138, 153, and 180. The high prevalence and magnitude of PCB 101 (greater than the limit of detection in >80% of the women in the cohort) are unique characteristics of this cohort. Great Lakes fish has been identified as a source of exposure to organochlorines in several studies. Spearman correlations and robust regression models were utilized to identify the impact of Great Lakes fish ingestion on cohort serum organochlorine concentrations. Several potential confounders of the relationship between serum organochlorines and Great Lakes fish consumption were identified. Covariates related to organochlorines in correlations as well as regression models included age, body surface area, fish ingestion, lipids, parity, race and smoking. Lower chlorinated PCB congeners do not follow the same trends as the higher chlorinated congeners and DDE. The higher chlorinated PCB congeners (PCB 138, 153, and 180) and DDE were correlated with age while the lower chlorinated congeners were not. PCB 153 and 180 regression models included age as a significant covariate. None of the higher chlorinated congeners correlated to race, while both lower chlorinated congeners were correlated to race. Race was also significant in both lower chlorinated congeners' regression models. PCB 101, a lower chlorinated congener seldom found in human serum, is readily found in the cohort. Airborne PCB exposure as well as diminished metabolism of PCB 101 in African Americans may explain the increased presence of PCB 101 and it's correlation with race. High end sport fish consumers (> or =1 meal per week) carried elevated levels of DDE and higher chlorinated PCB congeners (138, 153, and 180) compared to non-sport fish eaters. Unexpectedly, DDE was correlated more consistently with fish ingestion and age (a marker of bioaccumulation) in comparison to PCBs. Small correlations were found between serum PCBs and fish ingestion (Spearman correlation=0.19 for total PCBs and fish meals per year). Additionally, Serum PCBs in low end Great Lakes sport fish consumers were not higher than non-consumers. These findings suggest the women of the cohort are being exposed to PCBs through other routes in addition to Great lakes sport fish. One major route of exposure may be Chicago air. The observed trends amongst individual PCB congeners has important ramifications because lower chlorinated congeners or their metabolites may be mediators of toxicity. Organochlorine exposure through Great Lakes fish ingestion was clearly identified in high end fish consumers while associations with race, metabolism, and possible airborne exposures pose new questions.

The role of african american ethnicity and metabolism in sentinel polychlorinated biphenyl congener serum levels.

Polychlorinated biphenyls (PCBs) are environmental contaminants found in the serum of human populations across the globe. A small set of sentinel PCB congeners (IUPAC# 101, 118, 138, 153, and 180) commonly sought in human serum are often used as markers of exposure. The Chicago Great Lakes cohort of pregnant African American women was developed to study organochlorine exposure through Great Lakes resources in a pregnant African American population and their children. Comparison of PCB serum concentrations in women reporting mixed race/ethnicity within the cohort shows significant elevations of serum PCB 101 and 118 in women reporting exclusive African American ancestry.Incubations were performed using pooled human liver microsomes followed by individual recombinant human CYP isoform microsomes to identify whether the other sentinel congeners are metabolized by human CYP 450. In human liver microsome metabolism experiments with the sentinel PCB congeners (IUPAC# 101, 118, 138, 153, and 180), only PCB 101 metabolism produced an identifiable metabolite. However, a significant loss of parent compound was observed for PCB 118 incubations with human liver microsomes. The loss of PCB 101 and PCB 118 in microsome experiments indicates they are likely metabolized in human liver. Therefore, CYP 450 mediated metabolic differences may contribute to differences in serum concentrations by race/ethnicity.PCB metabolism has an important impact on toxicity. PCB metabolites have been shown to differ significantly in toxicity profiles relative to parent compounds. Biomonitoring studies of PCB serum levels have correlated with toxicity for the metabolizeable congeners such as PCB 101 and PCB 118. However, measureable amounts of metabolizeable parent congeners such as PCB 101 may not be detectable in the serum of study participants. Because PCB 118 is metabolized, but is also readily found in human serum, it may be a better marker of metabolism related PCB toxicity. Human specific PCB metabolism is difficult to characterize but has important pathophysiological ramifications and deserves further study.

Specific human CYP 450 isoform metabolism of a pentachlorobiphenyl (PCB-IUPAC# 101).

Polychlorinated biphenyl IUPAC# 101-PCB 101 (chlorination pattern-2,2',4',5,5') is a common, persistent non-coplanar PCB congener found in the ambient environment but information related to its metabolism in humans is lacking. Previous studies indicate PCB 101 is rapidly metabolized in mammals through CYP 2B and 3A family enzymes. Recently, PCB metabolism through a 2A family isoform in hamsters was also reported. To specifically identify the human CYP 450 isoforms responsible for PCB 101 metabolism, we compared human microsome metabolism to metabolism using several specific recombinant human CYP isoforms. These data characterized selective and extensive metabolism by human CYP 2A6. The product formed was the 4-hydroxy-PCB 101 metabolite (4-hydroxy-2,2',4',5,5') and was the only major metabolite observed in the recombinant and human microsome investigation. This is important information for predicting human specific toxicokinetics of PCBs.