Gordon P. McCallum

Oxidative Stress in Developmental Origins of Disease: Teratogenesis, Neurodevelopmental Deficits, and Cancer

In the developing embryo and fetus, endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS) like hydroxyl radicals may adversely alter development by oxidatively damaging cellular lipids, proteins and DNA, and/or by altering signal transduction. The postnatal consequences may include an array of birth defects (teratogenesis), postnatal functional deficits, and diseases. In animal models, the adverse developmental consequences of in utero exposure to agents like thalidomide, methamphetamine, phenytoin, benzo[a]pyrene, and ionizing radiation can be modulated by altering pathways that control the embryonic ROS balance, including enzymes that bioactivate endogenous substrates and xenobiotics to free radical intermediates, antioxidative enzymes that detoxify ROS, and enzymes that repair oxidative DNA damage. ROS-mediated signaling via Ras, nuclear factor kappa B and related transducers also may contribute to altered development. Embryopathies can be reduced by free radical spin trapping agents and antioxidants, and enhanced by glutathione depletion. Further modulatory approaches to evaluate such mechanisms in vivo and/or in embryo culture have included the use of knockout mice, transgenic knock-ins and mutant deficient mice with altered enzyme activities, as well as antisense oligonucleotides, protein therapy with antioxidative enzymes, dietary depletion of essential cofactors and chemical enzyme inhibitors. In a few cases, measures anticipated to be protective have conversely enhanced the risk of adverse developmental outcomes, indicating the complexity of development and need for caution in testing therapeutic strategies in humans. A better understanding of the developmental effects of ROS may provide insights for risk assessment and the reduction of adverse postnatal consequences.

Oxoguanine Glycosylase 1 Protects Against Methamphetamine-Enhanced Fetal Brain Oxidative DNA Damage and Neurodevelopmental Deficits

In utero methamphetamine (METH) exposure enhances the oxidative DNA lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in CD-1 fetal mouse brain, and causes long-term postnatal motor coordination deficits. Herein we used oxoguanine glycosylase 1 (ogg1) knock-out mice to determine the pathogenic roles of 8-oxoG and OGG1, which repairs 8-oxoG, in METH-initiated neurodevelopmental anomalies. Administration of METH (20 or 40 mg/kg) on gestational day 17 to pregnant +/− OGG1-deficient females caused a drug dose- and gene dose-dependent increase in 8-oxoG levels in OGG1-deficient fetal brains (p < 0.05). Female ogg1 knock-out offspring exposed in utero to high-dose METH exhibited gene dose-dependent enhanced motor coordination deficits for at least 12 weeks postnatally (p < 0.05). Contrary to METH-treated adult mice, METH-exposed CD-1 fetal brains did not exhibit altered apoptosis or DNA synthesis, and OGG1-deficient offspring exposed in utero to METH did not exhibit postnatal dopaminergic nerve terminal degeneration, suggesting different mechanisms. Enhanced 8-oxoG repair activity in fetal relative to adult organs suggests an important developmental protective role of OGG1 against in utero genotoxic stress. These observations provide the most direct evidence to date that 8-oxoG constitutes an embryopathic molecular lesion, and that functional fetal DNA repair protects against METH teratogenicity.

Oxidative DNA damage and repair in teratogenesis and neurodevelopmental deficits

Several teratogenic agents, including ionizing radiation and xenobiotics such as phenytoin, benzo[a]pyrene, thalidomide, and methamphetamine, can initiate the formation of reactive oxygen species (ROS) that oxidatively damage cellular macromolecules including DNA. Oxidative DNA damage, and particularly the most prevalent 8-oxoguanine lesion, may adversely affect development, likely via alterations in gene transcription rather than via a mutational mechanism. Contributions from oxidative DNA damage do not exclude roles for alternative mechanisms of initiation like receptor-mediated processes or the formation of covalent xenobiotic-macromolecular adducts, damage to other macromolecular targets like proteins and lipids, and other effects of ROS like altered signal transduction. Even in the absence of teratogen exposure, endogenous developmental oxidative stress can have embryopathic consequences in the absence of key pathways for detoxifying ROS or repairing DNA damage. Critical proteins in pathways for DNA damage detection/repair signaling, like p53 and ataxia telangiectasia mutated, and DNA repair itself, like oxoguanine glycosylase 1 and Cockayne syndrome B, can often, but not always, protect the embryo from ROS-initiating teratogens. Protection may be variably dependent upon such factors as the nature of the teratogen and its concentration within the embryo, the stage of development, the species, strain, gender, target tissue and cell type, among other factors.

Base excision repair of reactive oxygen species–initiated 7,8-dihydro-8-oxo-2′-deoxyguanosine inhibits the cytotoxicity of platinum anticancer drugs

Anticancer therapy with cisplatin and oxaliplatin is limited by toxicity and onset of tumor resistance. Both drugs form platinum-DNA cross-linked adducts, and cisplatin causes oxidative DNA damage including the 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) lesion. To assess oxidative DNA damage as a mechanism of cisplatin and oxaliplatin cytotoxicity, 8-oxodG–directed base excision repair was stably enhanced in human embryonic kidney cells by FLAG-tagged expression of human oxoguanine glycosylase 1 (α-OGG1) or its functional homologue, Escherichia coli formamidopyrimidine glycosylase (fpg). Both drugs increased reactive oxygen species and 8-oxodG levels, and cytotoxicity was decreased by antioxidant pretreatment. Ectopic expression of α-OGG1 or fpg in cell clones increased nuclear and mitochondrial 8-oxodG repair, and reduced death by reactive oxygen species initiators (H2O2, menadione) and both platinum drugs. Exposure to oxaliplatin caused a more marked and sustained block of cell proliferation than exposure to cisplatin. We conclude that the 8-oxodG lesion is cytotoxic, and base excision repair a likely determinant of risk. The greater antitumor efficacy of oxaliplatin seems unrelated to oxidative DNA damage, suggesting a novel strategy for improving the therapeutic index in cancer therapy. [Mol Cancer Ther 2009;8(7):2015–26]

Cockayne Syndrome B Protects Against Methamphetamine-Enhanced Oxidative DNA Damage in Murine Fetal Brain and Postnatal Neurodevelopmental Deficits

Methamphetamine (METH) increases the oxidative DNA lesion 8-oxoguanine (8-oxoG) in fetal mouse brain, and causes postnatal motor coordination deficits after in utero exposure. Like oxoguanine glycosylase 1 (OGG1), the Cockayne syndrome B (CSB) protein is involved in the repair of oxidatively damaged DNA, although its function is unclear. Here we used CSB-deficient Csb(m/m) knockout mice to investigate the developmental role of DNA oxidation and CSB in METH-initiated neurodevelopmental deficits. METH (40 mg/kg intraperitoneally) administration to pregnant Csb females on gestational day 17 increased 8-oxoG levels in Csb(m/m) fetal brains (p < 0.05). CSB modulated 8-oxoG levels independent of OGG1 activity, as 8-oxoG incision activity in fetal nuclear extracts was identical in Csb(m/m) and Csb(+/+)mice. This CSB effect was evident despite 7.1-fold higher OGG1 activity in Csb(+/+) mice compared to outbred CD-1 mice. Female Csb(m/m) offspring exposed in utero to METH exhibited motor coordination deficits postnatally (p < 0.05). In utero METH exposure did not cause dopaminergic nerve terminal degeneration, in contrast to adult exposures. This is the first evidence that CSB protects the fetus from xenobiotic-enhanced DNA oxidation and postnatal functional deficits, suggesting that oxidatively damaged DNA is developmentally pathogenic, and that fetal CSB activity may modulate the risk of reactive oxygen species-mediated adverse developmental outcomes.

Hypoxia Provokes Base Excision Repair Changes and a Repair-Deficient, Mutator Phenotype in Colorectal Cancer Cells

Regions of acute and chronic hypoxia exist within solid tumors and can lead to increased rates of mutagenesis and/or altered DNA damage and repair protein expression. Base excision repair (BER) is responsible for resolving small, non–helix-distorting lesions from the genome that potentially cause mutations by mispairing or promoting DNA breaks during replication. Germline and somatic mutations in BER genes, such as MutY Homolog (MUTYH/MYH) and DNA-directed polymerase (POLB), are associated with increased risk of colorectal cancer. However, very little is known about the expression and function of BER proteins under hypoxic stress. Using conditions of chronic hypoxia, decreased expression of BER proteins was observed because of a mechanism involving suppressed BER protein synthesis in multiple colorectal cancer cell lines. Functional BER was impaired as determined by MYH- and 8-oxoguanine (OGG1)–specific glycosylase assays. A formamidopyrimidine-DNA glycosylase (Fpg) Comet assay revealed elevated residual DNA base damage in hypoxic cells 24 hours after H2O2 treatment as compared with normoxic controls. Similarly, high-performance liquid chromatography analysis demonstrated that 8-oxo-2′-deoxyguanosine lesions were elevated in hypoxic cells 3 and 24 hours after potassium bromate (KBrO3) treatment when compared with aerobic cells. Correspondingly, decreased clonogenic survival was observed following exposure to the DNA base damaging agents H2O2 and MMS, but not to the microtubule interfering agent paclitaxel. Thus, a persistent downregulation of BER components by the microenvironment modifies and facilitates a mutator phenotype, driving genetic instability and cancer progression. Implications: Aberrant BER is a contributing factor for the observed genetic instability in hypoxic tumor cells. Mol Cancer Res; 12(10); 1407–15. ©2014 AACR.

Progression and metastasis in a transgenic mouse breast cancer model: Effects of exposure to in vivo hypoxia

Hypoxia is a predictor of poor patient survival in several cancers, including breast carcinomas. One possible mechanism is genomic instability induced by oxic stress. In this study we examined this possible mechanism by exposing an in vivo breast cancer model to hypoxia/reoxygenation. MMTV-Neu transgenic mice were exposed to cycling acute (AH) or chronic hypoxia (CH) before (early) or after (late) tumour detection to study effects of hypoxia on tumour initiation and progression, respectively. We observed no effect of the hypoxic exposures on times to first tumour detection, but we saw a trend of early AH-exposed mice to develop more tumours and macrometastases than CH-exposed mice. Unexpectedly, but consistent with these findings, we observed significantly reduced 8-oxo-dG lesions levels in the mammary tissue with the greatest difference observed between the air control (AC) and AH-exposed groups. In the late gassing group, there was a similar trend for reduced 8-oxo-dG lesion levels, but interestingly mice that developed macroscopic lung metastases demonstrated significantly increased levels of 8-oxo-dG lesions in their tumours relative to those that did not, irrespective of the gassing exposure. A trend for increased macrophage content was observed in tumours from mice exposed to acute hypoxia. Our results indicate that oxic stress induced by hypoxia/reoxygenation is unlikely to be a major factor driving tumour progression of established MMTV-Neu tumours but suggest that acute and chronic hypoxia may affect tumour incidence and metastasis when applied prior to tumour development.

Species differences in methanol and formic acid pharmacokinetics in mice, rabbits and primates

Methanol (MeOH) is metabolized primarily by alcohol dehydrogenase in humans, but by catalase in rodents, with species variations in the pharmacokinetics of its formic acid (FA) metabolite. The teratogenic potential of MeOH in humans is unknown, and its teratogenicity in rodents may not accurately reflect human developmental risk due to differential species metabolism, as for some other teratogens. To determine if human MeOH metabolism might be better reflected in rabbits than rodents, the plasma pharmacokinetics of MeOH and FA were compared in male CD-1 mice, New Zealand white rabbits and cynomolgus monkeys over time (24, 48 and 6h, respectively) following a single intraperitoneal injection of 0.5 or 2g/kg MeOH or its saline vehicle. Following the high dose, MeOH exhibited saturated elimination kinetics in all 3 species, with similar peak concentrations and a 2.5-fold higher clearance in mice than rabbits. FA accumulation within 6h in primates was 5-fold and 43-fold higher than in rabbits and mice respectively, with accumulation being 10-fold higher in rabbits than mice. Over 48 h, FA accumulation was nearly 5-fold higher in rabbits than mice. Low-dose MeOH in mice and rabbits resulted in similarly saturated MeOH elimination in both species, but with approximately 2-fold higher clearance rates in mice. FA accumulation was 3.8-fold higher in rabbits than mice. Rabbits more closely than mice reflected primates for in vivo MeOH metabolism, and particularly FA accumulation, suggesting that developmental studies in rabbits may be useful for assessing potential human teratological risk.

Methanol exposure does not lead to accumulation of oxidative DNA damage in bone marrow and spleen of mice, rabbits or primates.

Genotoxicity tests indicate methanol (MeOH) is not mutagenic, but a rodent study has suggested carcinogenic potential, which could result from free radical‐initiated oxidative DNA damage. To investigate this possibility we treated male CD‐1 mice, New Zealand white rabbits, and cynomolgus monkeys with MeOH (2.0 g/kg ip) and assessed tissue oxidative DNA damage 6 h post‐dose, measured as 8‐hydroxy‐2′‐deoxyguanosine (8‐oxodG). We found no MeOH‐dependent increases in 8‐oxodG in bone marrow or spleen of any species. Chronic treatment of CD‐1 mice with MeOH (2.0 g/kg ip) daily for 15 d also did not increase 8‐oxodG levels in these organs. Further studies in the DNA repair deficient oxoguanine glycosylase 1 (Ogg1) knockout (KO) mice supported these findings. Fibroblasts from Ogg1 KO mice accumulated 8‐oxodG following acute exposure to the renal carcinogen potassium bromate (KBrO3; 2.0 mM) but did not accumulate 8‐oxodG following exposure to 125 mM MeOH 6 h post‐treatment. Ogg1 KO mice accumulated 8‐oxodG in bone marrow and spleen with age but not following exposure to MeOH. In addition, free radical‐mediated hydroxynonenal‐histidine protein adducts were not enhanced by MeOH in primate bone marrow or spleen, or in rabbit bone marrow or mouse spleen, although modest increases were observed in rabbit spleen and mouse bone marrow. Taken together these observations suggest that MeOH exposure does not promote the accumulation of oxidative DNA damage in bone marrow and spleen, and it is unlikely that human environmental exposure to MeOH would lead to lymphomas via this mechanism. Mol. Carcinog.