Diverse mechanisms by which chemical pollutant exposure alters gut microbiota metabolism and inflammation.

The human gut microbiome, the host, and the environment are inextricably linked across the life course with significant health impacts. Consisting of trillions of bacteria, fungi, viruses, and other micro-organisms, microbiota living within our gut are particularly dynamic and responsible for digestion and metabolism of diverse classes of ingested chemical pollutants. Exposure to chemical pollutants not only in early life but throughout growth and into adulthood can alter human hosts' ability to absorb and metabolize xenobiotics, nutrients, and other components critical to health and longevity. Inflammation is a common mechanism underlying multiple environmentally related chronic conditions, including cardiovascular disease, multiple cancer types, and mental health. While growing research supports complex interactions between pollutants and the gut microbiome, significant gaps exist. Few reviews provide descriptions of the complex mechanisms by which chemical pollutants interact with the host microbiome through either direct or indirect pathways to alter disease risk, with a particular focus on inflammatory pathways. This review focuses on examples of several classes of pollutants commonly ingested by humans, including (i) heavy metals, (ii) persistent organic pollutants (POPs), and (iii) nitrates. Digestive enzymes and gut microbes are the first line of absorption and metabolism of these chemicals, and gut microbes have been shown to alter compounds from a less to more toxic state influencing subsequent distribution and excretion. In addition, chemical pollutants may interact with or alter the selection of more harmful and less commensal microbiota, leading to gut dysbiosis, and changes in receptor-mediated signaling pathways that alter the integrity and function of the gut intestinal tract. Arsenic, cadmium, and lead (heavy metals), influence the microbiome directly by altering different classes of bacteria, and subsequently driving inflammation through metabolite production and different signaling pathways (LPS/TLR4 or proteoglycan/TLR2 pathways). POPs can alter gut microbial composition either directly or indirectly depending on their ability to activate key signaling pathways within the intestine (e.g., PCB-126 and AHR). Nitrates and nitrites' effect on the gut and host may depend on their ability to be transformed to secondary and tertiary metabolites by gut bacteria. Future research should continue to support foundational research both in vitro, in vivo, and longitudinal population-based research to better identify opportunities for prevention, gain additional mechanistic insights into the complex interactions between environmental pollutants and the microbiome and support additional translational science.

Mice Lacking the Cytochrome P450 1B1 Gene Are Less Susceptible to Hyperoxic Lung Injury Than Wild Type.

Supplemental oxygen is a life-saving intervention administered to individuals suffering from respiratory distress, including adults with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Despite the clinical benefit, supplemental oxygen can create a hyperoxic environment that increases reactive oxygen species, oxidative stress, and lung injury. We have previously shown that cytochrome P450 (CYP)1A enzymes decrease susceptibility to hyperoxia-induced lung injury. In this investigation, we determined the role of CYP1B1 in hyperoxic lung injury in vivo. Eight- to ten-week old C57BL/6 wild type (WT) and Cyp1b1-/- mice were exposed to hyperoxia (>95% O2) for 24-72 h or maintained in room air (21% O2). Lung injury was assessed by histology and lung weight to body weight (LW/BW) ratios. Extent of inflammation was determined by assessing pulmonary neutrophil infiltration and cytokine levels. Lipid peroxidation markers were quantified by gas chromatography mass spectrometry, and oxidative DNA adducts were quantified by 32P-postlabeling as markers of oxidative stress. We found that Cyp1b1-/- mice displayed attenuation of lung weight and pulmonary edema, particularly after 48-72 h of hyperoxia compared with WT controls. Further, Cyp1b1-/- mice displayed decreased levels of pulmonary oxidative DNA adducts and pulmonary isofurans after 24 h of hyperoxia. Cyp1b1-/- mice also showed increased pulmonary CYP1A1 and 1A2 and mRNA expression. In summary, our results support the hypothesis that Cyp1b1-/- mice display decreased hyperoxic lung injury than wild type counterparts and that CYP1B1 may act as a pro-oxidant during hyperoxia exposure, contributing to increases in oxidative DNA damage and accumulation of lipid hydroperoxides.

ROLE OF CYTOCHROME P450S IN THE GENERATION AND METABOLISM OF REACTIVE OXYGEN SPECIES.

The cytochrome P450 (CYP) enzymes are a diverse group of heme monooxygenases that, through the course of their reaction cycle, contribute to cellular reactive oxygen species (ROS). CYP enzymes play a crucial role in human physiology and are involved in drug and xenobiotic metabolism as well as biosynthesis of endogenous molecules and are expressed throughout the human body. However, during the course of the CYP catalytic cycle, ROS can be generated through uncoupling of the enzymatic cycle. ROS is known to modify endogenous molecules, included lipids, proteins, and nucleic acids, which can lead to cell damage and death and contribute to disease development. ROS has been implicated in a wide range of diseases and conditions, including cancer and ageing, but ROS also play a role in the normal physiological functions in the cell. Here, we discuss specific examples whereby ROS generated by CYPs contribute to or protect against various phenomena, such as hyperoxic lung injury, oxidative hepatic toxicity, formation of DNA adducts from lipid peroxidation products. We have also discussed the mechanistic roles of CYP enzymes belonging to various families, and their effect on cellular ROS production, in relation to normal cellular function as well as disease pathophysiology.

Role of Cytochrome P450 (CYP)1A in Hyperoxic Lung Injury: Analysis of the Transcriptome and Proteome.

Hyperoxia contributes to lung injury in experimental animals and diseases such as acute respiratory distress syndrome in humans. Cytochrome P450 (CYP)1A enzymes are protective against hyperoxic lung injury (HLI). The molecular pathways and differences in gene expression that modulate these protective effects remain largely unknown. Our objective was to characterize genotype specific differences in the transcriptome and proteome of acute hyperoxic lung injury using the omics platforms: microarray and Reverse Phase Proteomic Array. Wild type (WT), Cyp1a1-/- and Cyp1a2-/- (8-10 wk, C57BL/6J background) mice were exposed to hyperoxia (FiO2 > 0.95) for 48 hours. Comparison of transcriptome changes in hyperoxia-exposed animals (WT versus knock-out) identified 171 genes unique to Cyp1a1-/- and 119 unique to Cyp1a2-/- mice. Gene Set Enrichment Analysis revealed pathways including apoptosis, DNA repair and early estrogen response that were differentially regulated between WT, Cyp1a1-/- and Cyp1a2-/- mice. Candidate genes from these pathways were validated at the mRNA and protein level. Quantification of oxidative DNA adducts with 32P-postlabeling also revealed genotype specific differences. These findings provide novel insights into mechanisms behind the differences in susceptibility of Cyp1a1-/- and Cyp1a2-/- mice to HLI and suggest novel pathways that need to be investigated as possible therapeutic targets for acute lung injury.

Mechanistic role of cytochrome P450 (CYP)1B1 in oxygen-mediated toxicity in pulmonary cells: A novel target for prevention of hyperoxic lung injury.

Supplemental oxygen, which is routinely administered to preterm infants with pulmonary insufficiency, contributes to bronchopulmonary dysplasia (BPD) in these infants. Hyperoxia also contributes to the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in adults. The mechanisms of oxygen-mediated pulmonary toxicity are not completely understood. Recent studies have suggested an important role for cytochrome P450 (CYP)1A1/1A2 in the protection against hyperoxic lung injury. The role of CYP1B1 in oxygen-mediated pulmonary toxicity has not been studied. In this investigation, we tested the hypothesis that CYP1B1 plays a mechanistic role in oxygen toxicity in pulmonary cells in vitro. In human bronchial epithelial cell line BEAS-2B, hyperoxic treatment for 1-3 days led to decreased cell viability by about 50-80%. Hyperoxic cytotoxicity was accompanied by an increase in levels of reactive oxygen species (ROS) by up to 110%, and an increase of TUNEL-positive cells by up to 4.8-fold. Western blot analysis showed hyperoxia to significantly down-regulate CYP1B1 protein level. Also, there was a decrease of CYP1B1 mRNA by up to 38% and Cyp1b1 promoter activity by up to 65%. On the other hand, CYP1B1 siRNA appeared to rescue the cell viability under hyperoxia stress, and overexpression of CYP1B1 significantly attenuated hyperoxic cytotoxicity after 48 h of incubation. In immortalized lung endothelial cells derived from Cyp1b1-null and wild-type mice, hyperoxia increased caspase 3/7 activities in a time-dependent manner, but endothelial cells lacking the Cyp1b1 gene showed significantly decreased caspase 3/7 activities after 48 and 72 h of incubation, implying that CYP1B1 might promote apoptosis in wild type lung endothelial cells under hyperoxic stress. In conclusion, our results support the hypothesis that CYP1B1 plays a mechanistic role in pulmonary oxygen toxicity, and CYP1B1-mediated apoptosis could be one of the mechanisms of oxygen toxicity. Thus, CYP1B1 could be a novel target for preventative and/or therapeutic interventions against BPD in infants and ALI/ARDS in adults.

U1A regulates 3' processing of the survival motor neuron mRNA.

Insufficient expression of the survival motor neuron (SMN) protein causes spinal muscular atrophy, a neurodegenerative disease characterized by loss of motor neurons. Despite the importance of maintaining adequate SMN levels, little is known about factors that control SMN expression, particularly 3' end processing of the SMN pre-mRNA. In this study, we identify the U1A protein as a key regulator of SMN expression. U1A, a component of the U1 snRNP, is known to inhibit polyadenylation upon direct binding to mRNA. We show that U1A binds directly and with high affinity and specificity to the SMN 3'-UTR adjacent to the polyadenylation site, independent of the U1 snRNP (U1 small nuclear ribonucleoprotein). Binding of U1A inhibits polyadenylation of the SMN pre-mRNA by specifically inhibiting 3' cleavage by the cleavage and polyadenylation specificity factor. Expression of U1A in excess of U1 snRNA causes inhibition of SMN polyadenylation and decreases SMN protein levels. This work reveals a new mechanism for regulating SMN levels and provides new insight into the roles of U1A in 3' processing of mRNAs.