Alcohol-induced CYP2E1, mitochondrial dynamics and retrograde signaling in human hepatic 3D organoids.

Alcohol toxicity is a significant health problem with ~3 million estimated deaths per year globally. Alcohol is metabolized to the toxic metabolite, acetaldehyde by alcohol dehydrogenase or CYP2E1 in the hepatic tissue, and also induces reactive oxygen species (ROS), which together play a pivotal role in cell and tissue damage. Our previous studies with COS-7 cells transduced with unique human CYP2E1 variants that mostly localize to either microsomes or mitochondria revealed that mitochondrially-localized CYP2E1 drives alcohol toxicity through the generation of higher levels of ROS, which has a consequent effect on cytochrome c oxidase (CcO) and mitochondrial oxidative function. Alcohol treatment of human hepatocyte cell line, HepaRG, in monolayer cultures increased ROS, affected CcO activity/stability, and induced mitophagy. Alcohol treatment of 3D organoids of HepaRG cells induced higher levels of CYP2E1 mRNA and activated mitochondrial stress-induced retrograde signaling, and also induced markers of hepatic steatosis. Knock down of CYP2E1 mRNA using specific shRNA, FK506, a Calcineurin inhibitor, and Mdivi-1, a DRP1 inhibitor, ameliorated alcohol-induced mitochondrial retrograde signaling, and hepatic steatosis. These results for the first time present a mechanistic link between CYP2E1 function and alcohol mediated mitochondrial dysfunction, retrograde signaling, and activation of hepatic steatosis in a 3D organoid system that closely recapitulates the in vivo liver response.

Mitochondria-targeted paraquat and metformin mediate ROS production to induce multiple pathways of retrograde signaling: A dose-dependent phenomenon.

The mitochondrial electron transport chain is a major source of reactive oxygen species (ROS) and is also a target of ROS, with an implied role in the stabilization of hypoxia-inducible factor (HIF) and induction of the AMPK pathway. Here we used varying doses of two agents, Mito-Paraquat and Mito-Metformin, that have been conjugated to cationic triphenylphosphonium (TPP+) moiety to selectively target them to the mitochondrial matrix compartment, thereby resulting in the site-specific generation of ROS within mitochondria. These agents primarily induce superoxide (O2•-) production by acting on complex I. In Raw264.7 macrophages, C2C12 skeletal myocytes, and HCT116 adenocarcinoma cells, we show that mitochondria-targeted oxidants can induce ROS (O2•- and H2O2). In all three cell lines tested, the mitochondria-targeted agents disrupted membrane potential and activated calcineurin and the Cn-dependent retrograde signaling pathway. Hypoxic culture conditions also induced Cn activation and HIF1α activation in a temporally regulated manner, with the former appearing at shorter exposure times. Together, our results indicate that mitochondrial oxidant-induced retrograde signaling is driven by disruption of membrane potential and activation of Ca2+/Cn pathway and is independent of ROS-induced HIF1α or AMPK pathways.

Mitochondrially targeted cytochrome P450 2D6 is involved in monomethylamine-induced neuronal damage in mouse models.

Parkinson's disease (PD) is a major human disease associated with degeneration of the central nervous system. Evidence suggests that several endogenously formed 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mimicking chemicals that are metabolic conversion products, especially ß-carbolines and isoquinolines, act as neurotoxins that induce PD or enhance progression of the disease. We have demonstrated previously that mitochondrially targeted human cytochrome P450 2D6 (CYP2D6), supported by mitochondrial adrenodoxin and adrenodoxin reductase, can efficiently catalyze the conversion of MPTP to the toxic 1-methyl-4-phenylpyridinium ion. In this study, we show that the mitochondrially targeted CYP2D6 can efficiently catalyze MPTP-mimicking compounds, i.e. 2-methyl-1,2,3,4-tetrahydroisoquinoline, 2-methyl-1,2,3,4-tetrahydro-ß-carboline, and 9-methyl-norharmon, suspected to induce PD in humans. Our results reveal that activity and respiration in mouse brain mitochondrial complex I are significantly affected by these toxins in WT mice but remain unchanged in Cyp2d6 locus knockout mice, indicating a possible role of CYP2D6 in the metabolism of these compounds both in vivo and in vitro These metabolic effects were minimized in the presence of two CYP2D6 inhibitors, quinidine and ajmalicine. Neuro-2a cells stably expressing predominantly mitochondrially targeted CYP2D6 were more sensitive to toxin-mediated respiratory dysfunction and complex I inhibition than cells expressing predominantly endoplasmic reticulum-targeted CYP2D6. Exposure to these toxins also induced the autophagic marker Parkin and the mitochondrial fission marker Dynamin-related protein 1 (Drp1) in differentiated neurons expressing mitochondrial CYP2D6. Our results show that monomethylamines are converted to their toxic cationic form by mitochondrially directed CYP2D6 and result in neuronal degradation in mice.

Cigarette Smoke Toxins-Induced Mitochondrial Dysfunction and Pancreatitis Involves Aryl Hydrocarbon Receptor Mediated Cyp1 Gene Expression: Protective Effects of Resveratrol.

We previously reported that mitochondrial CYP1 enzymes participate in the metabolism of polycyclic aromatic hydrocarbons and other carcinogens leading to mitochondrial dysfunction. In this study, using Cyp1b1-/-, Cyp1a1/1a2-/-, and Cyp1a1/1a2/1b1-/- mice, we observed that cigarette and environmental toxins, namely benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induce pancreatic mitochondrial respiratory dysfunction and pancreatitis. Our results suggest that aryl hydrocarbon receptor (AhR) activation and resultant mitochondrial dysfunction are associated with pancreatic pathology. BaP treatment markedly inhibits pancreatic mitochondrial oxygen consumption rate (OCR), ADP-dependent OCR, and also maximal respiration, in wild-type mice but not in Cyp1a1/1a2-/- and Cyp1a1/1a2/1b1-/- mice. In addition, both BaP and TCDD treatment markedly affected mitochondrial complex IV activity, in addition to causing marked reduction in mitochondrial DNA content. Interestingly, the AhR antagonist resveratrol, attenuated BaP-induced mitochondrial respiratory defects in the pancreas, and reversed pancreatitis, both histologically and biochemically in wild-type mice. These results reveal a novel role for AhR- and AhR-regulated CYP1 enzymes in eliciting mitochondrial dysfunction and cigarette toxin-mediated pancreatic pathology. We propose that increased mitochondrial respiratory dysfunction and oxidative stress are involved in polycyclic aromatic hydrocarbon associated pancreatitis. Resveratrol, a chemo preventive agent and AhR antagonist, and CH-223191, a potent and specific AhR inhibitor, confer protection against BaP-induced mitochondrial dysfunction and pancreatic pathology.

ß-Naphthoflavone-Induced Mitochondrial Respiratory Damage in Cyp1 Knockout Mouse and in Cell Culture Systems: Attenuation by Resveratrol Treatment.

A number of xenobiotic-inducible cytochrome P450s (CYPs) are now known to be localized in the mitochondrial compartment, though their pharmacological or toxicological roles remain unclear. Here, we show that BNF treatment markedly inhibits liver mitochondrial O2 consumption rate (OCR), ADP-dependent OCR, and also reserve OCR, in wild-type mice but not in Cyp1a1/1a2(-/-) double knockout mice. BNF treatment markedly affected mitochondrial complex I and complex IV activities and also attenuated mitochondrial gene expression. Furthermore, under in vitro conditions, BNF treatment induced cellular ROS production, which was inhibited by mitochondria-targeted antioxidant Mito-CP and CYP inhibitor proadefin, suggesting that most of the ROS production was intramitochondrial and probably involved the catalytic activity of mitochondrial CYP1 enzymes. Interestingly, our results also show that the AHR antagonist resveratrol, markedly attenuated BNF-induced liver mitochondrial defects in wild-type mice, confirming the role of AHR and AHR-regulated CYP1 genes in eliciting mitochondrial dysfunction. These results are consistent with reduced BNF-induced mitochondrial toxicity in Cyp1a1/1a2(-/-) mice and elevated ROS production in COS cells stably expressing CYP1A1. We propose that increased mitochondrial ROS production and respiratory dysfunction are part of xenobiotic toxicity. Resveratrol, a chemopreventive agent, renders protection against BNF-induced toxicity.

Mitochondrial targeting of cytochrome P450 (CYP) 1B1 and its role in polycyclic aromatic hydrocarbon-induced mitochondrial dysfunction.

We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41-48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria.

Autophagic vacuolation induced by excess ROS generation in HABP1/p32/gC1qR overexpressing fibroblasts and its reversal by polymeric hyaluronan.

The ubiquitous hyaladherin, hyaluronan-binding protein 1 (HABP1/p32/gC1qR) upon stable overexpression in normal fibroblasts (F-HABP07) has been reported to induce mitochondrial dysfunction, growth retardation and apoptosis after 72 h of growth. HABP1 has been observed to accumulate in the mitochondria resulting in generation of excess Reactive Oxygen Species (ROS), mitochondrial Ca(++) efflux and drop in mitochondrial membrane potential. In the present study, autophagic vacuolation was detected with monodansylcadaverin (MDC) staining from 36 h to 60 h of culture period along with elevated level of ROS in F-HABP07 cells. Increased expression of autophagic markers like MAP-LC3-II, Beclin 1 and autophagic modulator, DRAM confirmed the occurrence of the phenomenon. Reduced vacuole formation was observed upon treatment with 3-MA, a known PI3 kinase inhibitor, only at 32 h and was ineffective if treated later, as high ROS level was already attained. Treatment of F111 and F-HABP07 cells with bafilomycin A1 further indicated an increase in autophagosome formation along with autophagic degradation in HABP1 overexpressed fibroblasts. Comparison between normal fibroblast (F111) and F-HABP07 cells indicate reduced level of polymeric HA, its depolymerization and perturbed HA-HABP1 interaction in F-HABP07. Interestingly, supplementation of polymeric HA, an endogenous ROS scavenger, in the culture medium prompted reduction in number of vacuoles in F-HABP07 along with drop in ROS level, implying that excess ROS generation triggers initiation of autophagic vacuole formation prior to apoptosis due to overexpression of HABP1. Thus, the phenomenon of autophagy takes place prior to apoptosis induction in the HABP1 overexpressing cell line, F-HABP07.

Silencing of IkBß mRNA causes disruption of mitochondrial retrograde signaling and suppression of tumor growth in vivo.

A number of studies show that mitochondrial DNA (mtDNA) depletion and attendant activation of retrograde signaling induces tumor progression. We have reported previously that activation of a novel nuclear factor-Kappa B pathway is critical for the propagation of mitochondrial retrograde signaling, which induces both phenotypic and morphological changes in C2C12 myoblasts and A549 lung carcinoma cells. In this study, we investigated the role of stress-induced nuclear factor-Kappa B in tumor progression in xenotransplanted mice. We used a retroviral system for the inducible expression of small interfering RNA against IkBα and IkBß mRNAs. Expression of small interfering RNA against IkBß markedly impaired tumor growth and invasive ability of mtDNA-depleted C2C12 myoblasts and also thwarted anchorage-independent growth of the cells. Knockdown of IkBα mRNA, however, did not have any modulatory effect in this cell system. Moreover, expression of small interfering RNA against IkBß reduced the expression of marker genes for retrograde signaling and tumor growth in xenografts of mtDNA-depleted cells. Our findings demonstrate that IkBß is a master regulator of mitochondrial retrograde signaling pathway and that the retrograde signaling plays a role in tumor growth in vivo. In this regard, IkBß supports the tumorigenic potential of mtDNA-depleted C2C12 cells.

Additive effects of mitochondrion-targeted cytochrome CYP2E1 and alcohol toxicity on cytochrome c oxidase function and stability of respirosome complexes.

Alcohol treatment induces oxidative stress by a combination of increased production of partially reduced oxygen species and decreased cellular antioxidant pool, including GSH. Recently, we showed that mitochondrion-targeted CYP2E1 augments alcohol-mediated toxicity, causing an increase in reactive oxygen species production and oxidative stress. Here, we show that cytochrome c oxidase (CcO), the terminal oxidase of the mitochondrial respiratory chain, is a critical target of CYP2E1-mediated alcohol toxicity. COS-7 and Hep G2 cell lines expressing predominantly mitochondrion-targeted (Mt(++)) CYP2E1 and livers from alcohol-treated rats showed loss of CcO activity and increased protein carbonylation, which was accompanied by a decline in the steady state levels of subunits I, IVI1, and Vb of the CcO complex. This was also accompanied by reduced mitochondrial DNA content and reduced mitochondrial mRNA. These changes were more prominent in Mt(++) cells in comparison with wild type (WT) CYP2E1-expressing or ER(+) (mostly microsome-targeted) cells. In addition, mitochondrion-specific antioxidants, ubiquinol conjugated to triphenyl phosphonium, triphenylphosphonium conjugated carboxyl proxyl, and the CYP2E1 inhibitor diallyl sulfide prevented the loss of CcO activity and the CcO subunits, most likely through reduced oxidative damage to the enzyme complex. Our results suggest that damage to CcO and dissociation of respirosome complexes are critical factors in alcohol-induced toxicity, which is augmented by mitochondrion-targeted CYP2E1. We propose that CcO is one of the direct and immediate targets of alcohol-induced toxicity causing respiratory dysfunction.

Evidence for Serpentine as a novel antioxidant by a redox sensitive HABP1 overexpressing cell line by inhibiting its nuclear translocation of NF-κB.

Herbal antioxidants are gradually gaining importance as dietary supplements considering the growing implications of oxidative stress in most degenerative diseases and aging. Thus, continuous attempts are made to search for novel herbal molecules with antioxidative properties, using chemical methods predominantly with the need arising for cell based assays. We have generated a stable cell line F-HABP07, by constitutively overexpressing human Hyaluronan Binding Protein1 (HABP1) in murine fibroblasts which accumulates in the mitochondria leading to excess ROS generation without any external stimuli. In the present study, we demonstrated the nuclear translocation of p65 subunit of NF-κB in F-HABP07 cells, an important signature of ROS induced signalling cascade providing us an opportunity to use it as a screening system for ROS scavengers. Using known antioxidants on our designer cell line, we have demonstrated a dose dependant reduction in ROS generation and observed inhibition of p65 subunit of NF-κB nuclear translocation, increase in glutathione content and down-regulation of apoptotic marker Bax establishing its antioxidant biosensing capacity. With the help of this cell line, we for the first time demonstrated serpentine, one of the active components from the roots of Rauwolfia serpentina (a traditional medicinal plant), to be a novel non-cytotoxic antioxidant. The authenticity of this cell line screening system based discovery was validated using standard chemical assays thus, opening up new therapeutic avenues for this herbal compound and the use of this designer cell line.

Excessive reactive oxygen species induces apoptosis in fibroblasts: role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR).

Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities along with initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca 2+ influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.

Differential expression of Hyaluronic Acid Binding Protein 1 (HABP1)/P32/C1QBP during progression of epidermal carcinoma.

Our laboratory has characterized a novel cell surface glycoprotein, Hyaluronic Acid Binding Protein 1 (HABP1), interacting specifically with hyaluronan (HA) and regulating HA-mediated cellular event. The involvement of HA in different stages of carcinoma is well documented. In the present communication, the expression profile of HABP1 was investigated from initiation to progression of epidermal carcinoma in mice, induced by benzo[a]pyrene (B[a]P) exposure. During tumor initiation, HABP1 accumulated in inflammatory subsquamous tissue and with progression, the protein, was also seen to overexpress in papillomatic and acanthotic tissue. With the onset of metastasis, HABP1 overexpression was confined to metastatic islands, while it disappeared gradually from the surrounding mass. Such expression profiles in metastasized tissue were supported by decreased levels of HABP1, both at protein and transcript levels. These observations taken together suggest that the changes in HABP1 level coincide with specific stages of tumor progression, that lead to disruption of its interaction with HA, implying a role in the regulation of tumor metastasis.