Michal W. Luczak

Role of direct reactivity with metals in chemoprotection by N-acetylcysteine against chromium(VI), cadmium(II), and cobalt(II)

The antioxidant N-acetylcysteine (NAC) is widely used for the assessment of the role of reactive oxygen species (ROS) in various biological processes and adverse drug reactions. NAC has been found to effectively inhibit the toxicity of carcinogenic metals, which was attributed to its potent ROS-suppressive properties. However, the absence of redox activity among some metals and findings from genetic models suggested a more diverse, smaller role of oxidative stress in metal toxicity. Here, we examined mechanisms of chemoprotection by NAC against Cd(II), Co(II), and Cr(VI) in human cells. We found that NAC displayed a broad-spectrum chemoprotective activity against all three metals, including suppression of cytotoxicity, apoptosis, p53 activation, and HSP72 and HIF-1α upregulation. Cytoprotection by NAC was independent of cellular glutathione. NAC strongly inhibited the uptake of all three metals in histologically different types of human cells, explaining its high chemoprotective potential. A loss of Cr(VI) accumulation by cells was caused by NAC-mediated extracellular reduction of chromate to membrane-impermeative Cr(III). Suppression of Co(II) uptake resulted from a rapid formation of Co(II)-NAC conjugates that were unable to enter cells. Our results demonstrate that NAC acts through more than one mechanism in preventing metal toxicity and its chemoprotective activity can be completely ROS-independent. Good clinical safety and effectiveness in Co(II) sequestration suggest that NAC could be useful in the prevention of tissue accumulation and toxic effects of Co ions released by cobalt-chromium hip prostheses.

Pharmacokinetic studies of enantiomers of ibuprofen and its chiral metabolites in humans with different variants of genes coding CYP2C8 and CYP2C9 isoenzymes

The pharmacokinetics of ibuprofen enantiomers and its chiral metabolites, namely (R,S)-29-hydroxyibuprofen and (RR,RS,SR,SS)-29-carboxyibuprofen, was studied in healthy volunteers carrying different alleles coding cytochrome P450 (CYP) 4502C isoenzymes. Following administration of  400 mg of racemic ibuprofen, enantiomers of the parent compound and their metabolites were isolated from plasma and urine samples using solid-phase extraction and were quantified by the validated capillary zone electrophoresis method. The levels of the analytes in biological fluids were used to calculate their pharmacokinetic parameters in subjects with different variants of CYP2C8 and CYP2C9 isoenzymes. The analysis of each subject’s genotype was carried out using polymerase chain reaction-restriction fragment length polymorphism. Impaired metabolism of ibuprofen enantiomers was associated with the presence of CYP2C8*3, CYP2C9*2 and CYP2C9*3 alleles. The greatest effect of mutated alleles on pharmacokinetics was observed in a subject with a CYP2C8*1/*3, CYP2C9*1/*2 genotype. This subject appeared to have lower value of clearance, greater area under the curve (AUC) and longer time t0.5 in comparison with the wild-type.

Uptake, p53 Pathway Activation, and Cytotoxic Responses for Co(II) and Ni(II) in Human Lung Cells: Implications for Carcinogenicity

Cobalt(II) and nickel(II) ions display similar chemical properties and act as hypoxia mimics in cells. However, only soluble Co(II) but not soluble Ni(II) is carcinogenic by inhalation. To explore potential reasons for these differences, we examined responses of human lung cells to both metals. We found that Co(II) showed almost 8 times higher accumulation than Ni(II) in H460 cells but caused a less efficient activation of the transcriptional factor p53 as measured by its accumulation, Ser15 phosphorylation, and target gene expression. Unlike Ni(II), Co(II) was ineffective in downregulating the p53 inhibitor MDM4 (HDMX). Co(II)-treated cells continued DNA replication at internal doses that caused massive apoptosis by Ni(II). Apoptosis and the overall cell death by Co(II) were delayed and weaker than by Ni(II). Inhibition of caspases but not programmed necrosis pathways suppressed Co(II)-induced cell death. Knockdown of p53 produced 50%-60% decreases in activation of caspases 3/7 and expression of 2 most highly upregulated proapoptotic genes PUMA and NOXA by Co(II). Overall, p53-mediated apoptosis accounted for 55% cell death by Co(II), p53-independent apoptosis for 20%, and p53/caspase-independent mechanisms for 25%. Similar to H460, normal human lung fibroblasts and primary human bronchial epithelial cells had several times higher accumulation of Co(II) than Ni(II) and showed a delayed and weaker caspase activation by Co(II). Thus, carcinogenicity of soluble Co(II) could be related to high survival of metal-loaded cells, which permits accumulation of genetic and epigenetic abnormalities. High cytotoxicity of soluble Ni(II) causes early elimination of damaged cells and is expected to be cancer suppressive.

Expression of HOXA-10 and HOXA-11 in the endometria of women with idiopathic infertility

In fertile women, HOXA-10 and HOXA-11 expression rises during the luteal phase, with the peak occurring during the implantation window, and stays at a high level until the end of the cycle. We evaluated the transcript and protein levels of HOXA-10 and HOXA-11 in the endometria of patients with idiopathic infertility (n = 15) and control patients (n = 10). The amounts of mRNA were determined by reverse transcription and real-time quantitative PCR. The protein levels were evaluated by Western blotting analysis. Using immunohistochemical techniques, we compared the localization of HOXA-10 and HOXA-11 proteins in the implantation window between the study and control groups. We observed statistically significantly decreased HOXA-10 and HOXA-11 transcript levels (p = 0.003, p = 0.012 respectively) in infertile patients compared to controls. There was no significant decrease in HOXA-10 protein levels between these groups (p = 0.074). However, we observed a significantly higher level of HOXA-11 protein in the endometria of infertile patients compared to controls (p = 0.015). HOXA-10 and HOXA-11 proteins were localized in the nuclei of the endometrial stromal cells. Immunohistochemical analyses did not reveal differences between amounts of HOXA-10 and HOXA-11 protein levels in infertility and control groups. Our results suggest that HOXA-10 and HOXA-11 gene expression in the endometrium during the implantation window may not be altered in patients with idiopathic infertility.

Monitoring Cr intermediates and reactive oxygen species with fluorescent probes during chromate reduction.

Cr(VI) genotoxicity is caused by products of its reductive metabolism inside the cells. Reactive oxygen species (ROS) and Cr(V,IV) intermediates are potential sources of oxidative damage by Cr(VI). Here, we investigated seven fluorescent probes for the detection of ROS and non-ROS oxidants in Cr(VI) reactions with its main reducers. We found that Cr(V)-skipping metabolism of Cr(VI) by ascorbate in vitro gave no responses with all tested dyes, indicating nonreactivity of Cr(IV) and absence of ROS. Cr(VI) reduction with glutathione (GSH) or Cys strongly enhanced the fluorescence of dichlorofluorescein (DCF) and dihydrorhodamine 123 (DHR123) but produced minimal fluorescence with dihydroethidium and no increases with aminophenylfluorescein and CellRox Green, Orange, and Red. Several tests showed that Cr(VI)-thiol reactions lacked ROS and that Cr(V) caused oxidation of DCF and DHR123. DCF reacted only with free Cr(V), whereas DHR123 detected both the free Cr(V) and Cr(V)-GSH complex. We estimated that Cr(VI)-GSH reactions generated approximately 75% Cr(V)-GSH and 25% free Cr(V), whereas Cys reactions appeared to produce only free Cr(V). DHR123 measurements in H460 cells showed that reduction of Cr(VI) was complete within 20 min postexposure, but it lasted at least 1 h without GSH. Cells with restored ascorbate levels exhibited no DCF or DHR123 oxidation by Cr(VI). Overall, our results demonstrated that Cr(VI) metabolism with its biological reducers lacked ROS and that DHR123 and DCF responses were indicators of total and free Cr(V), respectively. CellRox dyes, dihydroethidium and aminophenylfluorescein, are insensitive to Cr(V,IV) and can be used for monitoring ROS during coexposure to Cr(VI) and oxidants.

Chromium(VI) Causes Interstrand DNA Cross-Linking in Vitro but Shows No Hypersensitivity in Cross-Link Repair-Deficient Human Cells

Hexavalent chromium is a human carcinogen activated primarily by direct reduction with cellular ascorbate and to a lesser extent, by glutathione. Cr(III), the final product of Cr(VI) reduction, forms six bonds allowing intermolecular cross-linking. In this work, we investigated the ability of Cr(VI) to cause interstrand DNA cross-links (ICLs) whose formation mechanisms and presence in human cells are currently uncertain. We found that in vitro reduction of Cr(VI) with glutathione showed a sublinear production of ICLs, the yield of which was less than 1% of total Cr-DNA adducts at the optimal conditions. Formation of ICLs in fast ascorbate-Cr(VI) reactions occurred during a short reduction interval and displayed a linear dose dependence with the average yield of 1.3% of total adducts. In vitro production of ICLs was strongly suppressed by increasing buffer molarity, indicating inhibitory effects of ligand-Cr(III) binding on the formation of cross-linking species. The presence of ICLs in human cells was assessed from the impact of ICL repair deficiencies on Cr(VI) responses. We found that ascorbate-restored FANCD2-null and isogenic FANCD2-complemented cells showed similar cell cycle inhibition and toxicity by Cr(VI). XPA-null cells are defective in the repair of Cr-DNA monoadducts, but stable knockdowns of ERCC1 or XPF in these cells with extended time for the completion of cross-linking reactions did not produce any sensitization to Cr(VI). Our results together with chemical and steric considerations of Cr(III) reactivity suggest that ICL generation by chromate is probably an in vitro phenomenon occurring at conditions permitting the formation of Cr(III) oligomers.

ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA.

Different ATM Signaling in Response to Chromium(VI) Metabolism via Ascorbate and Nonascorbate Reduction: Implications for in Vitro Models and Toxicogenomics.

Background Carcinogenic hexavalent chromium [Cr(VI)] requires cellular reduction to generate DNA damage. Metabolism of Cr(VI) by its principal reducer ascorbate (Asc) lacks a Cr(V) intermediate, which is abundant in reactions with a minor reducing agent, glutathione. Cultured cells are widely used in mechanistic studies of Cr(VI) toxicity; however, they typically contain < 1% of normal Asc levels. Asc deficiency is also expected to diminish protection against reactive oxygen species. Objectives We assessed how the presence of Asc in cells affects their stress signaling and survival responses to chromate. Methods We investigated the effects of Asc restoration in human lung H460 cells and normal human lung fibroblasts on the activation and functional role of ATM kinase, which controls DNA damage responses involving several hundreds of proteins. Results Treatment of standard cultures with Cr(VI) strongly activated ATM, as indicated by its automodification at Ser1981 and by phosphorylation of checkpoint kinase 2 (CHK2) and chromatin/transcription regulator KRAB-associated protein 1 (KAP1). Confirming the importance of activated ATM, its inhibition impaired replication recovery and clonogenic survival. In contrast, fully Asc-restored cells lacked ATM activation by Cr(VI), and ATM silencing produced no significant effects on p53 stabilization, apoptosis, replication recovery, or clonogenic survival. Dose dependence studies found a close correlation between ATM activation and the extent of Cr(VI) reduction by glutathione. Conclusions Asc restoration in cultured cells dramatically altered their stress responses to Cr(VI) by preventing activation of the oxidant-sensitive ATM network. We suggest that toxicogenomic and other cell response-based approaches likely underestimate Cr(VI) genotoxicity when standard ATM-activating carcinogens are used as references. Citation Luczak MW, Green SE, Zhitkovich A. 2016. Different ATM signaling in response to chromium(VI) metabolism via ascorbate and nonascorbate reduction: implications for in vitro models and toxicogenomics. Environ Health Perspect 124:61–66; http://dx.doi.org/10.1289/ehp.1409434

Nickel-induced HIF-1α promotes growth arrest and senescence in normal human cells but lacks toxic effects in transformed cells

&NA; Nickel is a human carcinogen that acts as a hypoxia mimic by activating the transcription factor HIF‐1&agr; and hypoxia‐like transcriptomic responses. Hypoxia and elevated HIF‐1&agr; are typically associated with drug resistance in cancer cells, which is caused by increased drug efflux and other mechanisms. Here we examined the role of HIF‐1&agr; in uptake of soluble Ni(II) and Ni(II)‐induced cell fate outcomes using si/shRNA knockdowns and gene deletion models. We found that HIF‐1&agr; had no effect on accumulation of Ni(II) in two transformed (H460, A549) and two normal human cell lines (IMR90, WI38). The loss of HIF‐1&agr; also produced no significant impact on p53‐dependent and p53‐independent apoptotic responses or clonogenic survival of Ni(II)‐treated transformed cells. In normal human cells, HIF‐1&agr; enhanced the ability of Ni(II) to inhibit cell proliferation and cause a permanent growth arrest (senescence). Consistent with its growth‐suppressive effects, HIF‐1&agr; was important for upregulation of the cell cycle inhibitors p21 (CDKN1A) and p27 (CDKN1B). Irrespective of HIF‐1&agr; status, Ni(II) strongly increased levels of MYC protein but did not change protein expression of the cell cycle‐promoting phosphatase CDC25A or the CDK inhibitor p16. Our findings indicate that HIF‐1&agr; limits propagation of Ni(II)‐damaged normal cells, suggesting that it may act in a tumor suppressor‐like manner during early stages of Ni(II) carcinogenesis. HighlightsHIF‐1&agr; enhances growth arrest and senescence by Ni(II) in normal human cells.HIF‐1&agr; is important for upregulation of the cell cycle inhibitors p21 and p27 in Ni(II)‐treated normal cells.Apoptotic responses and clonogenic toxicity by Ni(II) in transformed human cells are HIF‐1&agr; independent.HIF‐1&agr; does not affect Ni(II) accumulation in normal or transformed human cells.

Monoubiquitinated γ-H2AX: Abundant product and specific biomarker for non-apoptotic DNA double-strand breaks

ABSTRACT DNA double‐strand breaks (DSBs) are a highly toxic form of DNA damage produced by a number of carcinogens, drugs, and metabolic abnormalities. Involvement of DSBs in many pathologies has led to frequent measurements of these lesions, primarily via biodosimetry of S139‐phosphorylated histone H2AX (&ggr;‐H2AX). However, &ggr;‐H2AX is also induced by some non‐DSB conditions and abundantly formed in apoptosis, raising concerns about the overestimation of potential genotoxic agents and accuracy of DSB assessments. DSB‐triggered &ggr;‐H2AX undergoes RNF168‐mediated K13/K15 monoubiquitination, which is rarely analyzed in DSB/genotoxicity studies. Here we identified critical methodological factors that are necessary for the efficient detection of mono‐ (ub1) and diubiquitinated (ub2) &ggr;‐H2AX. Using optimized technical conditions, we found that &ggr;‐H2AX‐ub1 was a predominant form of &ggr;‐H2AX in three primary human cell lines containing mechanistically distinct types of DSBs. Replication stress‐associated DSBs also triggered extensive formation of &ggr;‐H2AX‐ub1. For DSBs induced by oxidative damage or topoisomerase II, both &ggr;‐H2AX and &ggr;‐H2AX‐ub1 showed dose‐dependent increases whereas &ggr;‐H2AX‐ub2 plateaued at low levels of breaks. Despite abundance of &ggr;‐H2AX, &ggr;‐H2AX‐ub1,2 formation was blocked in apoptosis, which was associated with proteolytic cleavage of RNF168. Chromatin damage also caused only the production of &ggr;‐H2AX but not its ub1,2 forms. Our results revealed a major contribution of ubiquitinated forms to the overall &ggr;‐H2AX response and demonstrated the specificity of monoubiquitinated &ggr;‐H2AX as a biodosimeter of non‐apoptotic DSBs. HIGHLIGHTSOptimized conditions for detection of monoubiquitinated &ggr;‐H2AX (&ggr;‐H2AX‐ub1)&ggr;‐H2AX‐ub1 is a main form in normal cells with DNA double‐strand breaks (DSBs)&ggr;‐H2AX‐ub1 is a dose‐dependent biomarker of DSBs arising as DNA damageUnlike &ggr;‐H2AX, &ggr;‐H2AX‐ub1 was absent in apoptotic cells or after chromatin damage