Effect of Vitamin C Infusion on Organ Failure and Biomarkers of Inflammation and Vascular Injury in Patients With Sepsis and Severe Acute Respiratory Failure: The CITRIS-ALI Randomized Clinical Trial.

Importance: Experimental data suggest that intravenous vitamin C may attenuate inflammation and vascular injury associated with sepsis and acute respiratory distress syndrome (ARDS). Objective: To determine the effect of intravenous vitamin C infusion on organ failure scores and biological markers of inflammation and vascular injury in patients with sepsis and ARDS. Design, Setting, and Participants: The CITRIS-ALI trial was a randomized, double-blind, placebo-controlled, multicenter trial conducted in 7 medical intensive care units in the United States, enrolling patients (N = 167) with sepsis and ARDS present for less than 24 hours. The study was conducted from September 2014 to November 2017, and final follow-up was January 2018. Interventions: Patients were randomly assigned to receive intravenous infusion of vitamin C (50 mg/kg in dextrose 5% in water, n = 84) or placebo (dextrose 5% in water only, n = 83) every 6 hours for 96 hours. Main Outcomes and Measures: The primary outcomes were change in organ failure as assessed by a modified Sequential Organ Failure Assessment score (range, 0-20, with higher scores indicating more dysfunction) from baseline to 96 hours, and plasma biomarkers of inflammation (C-reactive protein levels) and vascular injury (thrombomodulin levels) measured at 0, 48, 96, and 168 hours. Results: Among 167 randomized patients (mean [SD] age, 54.8 years [16.7]; 90 men [54%]), 103 (62%) completed the study to day 60. There were no significant differences between the vitamin C and placebo groups in the primary end points of change in mean modified Sequential Organ Failure Assessment score from baseline to 96 hours (from 9.8 to 6.8 in the vitamin C group [3 points] and from 10.3 to 6.8 in the placebo group [3.5 points]; difference, -0.10; 95% CI, -1.23 to 1.03; P = .86) or in C-reactive protein levels (54.1 vs 46.1 µg/mL; difference, 7.94 µg/mL; 95% CI, -8.2 to 24.11; P = .33) and thrombomodulin levels (14.5 vs 13.8 ng/mL; difference, 0.69 ng/mL; 95% CI, -2.8 to 4.2; P = .70) at 168 hours. Conclusions and Relevance: In this preliminary study of patients with sepsis and ARDS, a 96-hour infusion of vitamin C compared with placebo did not significantly improve organ dysfunction scores or alter markers of inflammation and vascular injury. Further research is needed to evaluate the potential role of vitamin C for other outcomes in sepsis and ARDS. Trial Registration: ClinicalTrials.gov Identifier: NCT02106975.

Development of a goat model for evaluation of withaferin A: Cervical implants for the treatment of cervical intraepithelial neoplasia.

Cervical cancer is caused by human papillomavirus (HPV). The disease develops over many years through a series of precancerous lesions. Cervical cancer can be prevented by HPV-vaccination, screening and treatment of precancer before development of cervical cancer. The treatment of high-grade cervical dysplasia (CIN 2+) has traditionally been by cervical conization. Surgical procedures are associated with increased risk of undesirable side effects including bleeding, infection, scarring (stenosis), infertility and complications in later pregnancies. An inexpensive, non-invasive method of delivering therapeutics locally will be favorable to treat precancerous cervical lesions without damaging healthy tissue. The feasibility and safety of a sustained, continuous drug-releasing cervical polymeric implant for use in clinical trials was studied using a large animal model. The goat (Capra hircus), non-pregnant adult female Boer goats, was chosen due to similarities in cervical dimensions to the human. Estrus was induced with progesterone CIDR® vaginal implants for 14days followed by the administration of chorionic gonadotropins 48h prior to removal of the progesterone implants to relax the cervix to allow for the placement of the cervical implant. Cervical implants, containing 2% and 4% withaferin A (WFA), with 8 coats of blank polymer, provided sustained release for a long duration and were used for the animal study. The 'mushroom'-shaped cervical polymeric implant, originally designed for women required redesigning to be accommodated within the goat cervix. The cervical implants were well tolerated by the animals with no obvious evidence of discomfort, systemic or local inflammation or toxicity. In addition, we developed a new method to analyze tissue WFA levels by solvent extractions and LS/MS-MS. WFA was found to be localized to the target and adjacent tissues with 12-16ng WFA/g tissue, with essentially no detectable WFA in distant tissues. This study suggests that the goat is a good large animal model for the future development and evaluation of therapeutic efficacy of continuous local drug delivery by cervical polymeric implants to treat precancerous cervical lesions.

Milk-derived exosomes for oral delivery of paclitaxel.

In this report milk-derived exosomes have been investigated for oral delivery of the chemotherapeutic drug paclitaxel (PAC) as an alternative to conventional i.v. therapy for improved efficacy and reduced toxicity. PAC-loaded exosomes (ExoPAC) were found to have a particle size of ~108 nm, a narrow particle size distribution (PDI ~0.190), zeta potential (~ -7 mV) and a practical loading efficiency of ~8%. Exosomes and ExoPAC exhibited excellent stability in the presence of simulated-gastrointestinal fluids, and during the storage at -80 °C. A sustained release of PAC was also observed up to 48 h in vitro using PBS (pH 6.8). Importantly, ExoPAC delivered orally showed significant tumor growth inhibition (60%; P<0.001) against human lung tumor xenografts in nude mice. Treatment with i.p. PAC at the same dose as ExoPAC, however, showed modest but statistically insignificant inhibition (31%). Moreover, ExoPAC demonstrated remarkably lower systemic and immunologic toxicities as compared to i.v. PAC.

Injuries Reported and Recorded for Adults with Intellectual Disabilities Who Live with Paid Support in Scotland: a Comparison with Scottish Adults in the General Population.

BACKGROUND: Providers of supported living services to adults with intellectual disabilities (IDs) in the United Kingdom have procedures in place to monitor injuries; this provides opportunity to learn about the injuries being reported and recorded. The aim was to determine the incidence, causes and types of injuries experienced by 593 adults with intellectual disabilities who live with paid support in a 12-month period. METHOD: Injury data, collected via a standard electronic injury monitoring system, were compared with data collected for a matched sample of the general population in the same year. RESULTS: The adults with intellectual disabilities experienced a higher rate of injury. Falls were the commonest cause of injury for both samples, but significantly more so for the adults with intellectual disabilities. CONCLUSIONS: The higher rate of injuries, particularly minor injuries, being reported suggests a culture of injury reporting and recording within these supported living services. Electronic injury monitoring is recommended for organizations providing supported living services for adults with intellectual disabilities.

Exosomes as an Emerging Plasmid Delivery Vehicle for Gene Therapy.

Despite its introduction more than three decades ago, gene therapy has fallen short of its expected potential for the treatment of a broad spectrum of diseases and continues to lack widespread clinical use. The fundamental limitation in clinical translatability of this therapeutic modality has always been an effective delivery system that circumvents degradation of the therapeutic nucleic acids, ensuring they reach the intended disease target. Plasmid DNA (pDNA) for the purpose of introducing exogenous genes presents an additional challenge due to its size and potential immunogenicity. Current pDNA methods include naked pDNA accompanied by electroporation or ultrasound, liposomes, other nanoparticles, and cell-penetrating peptides, to name a few. While the topic of numerous reviews, each of these methods has its own unique set of limitations, side effects, and efficacy concerns. In this review, we highlight emerging uses of exosomes for the delivery of pDNA for gene therapy. We specifically focus on bovine milk and colostrum-derived exosomes as a nano-delivery "platform". Milk/colostrum represents an abundant, scalable, and cost-effective natural source of exosomes that can be loaded with nucleic acids for targeted delivery to a variety of tissue types in the body. These nanoparticles can be functionalized and loaded with pDNA for the exogenous expression of genes to target a wide variety of disease phenotypes, overcoming many of the limitations of current gene therapy delivery techniques.

Milk/colostrum exosomes: A nanoplatform advancing delivery of cancer therapeutics.

Chemotherapeutics continue to play a central role in the treatment of a wide variety of cancers. Conventional chemotherapy involving bolus intravenous doses results in severe side effects - in some cases life threatening - delayed toxicity and compromised quality-of-life. Attempts to deliver small drug molecules using liposomes, polymeric nanoparticles, micelles, lipid nanoparticles, etc. have produced limited nanoformulations for clinical use, presumably due to a lack of biocompatibility of the material, costs, toxicity, scalability, and/or lack of effective administration. Naturally occurring small extracellular vesicles, or exosomes, may offer a solution and a viable system for delivering cancer therapeutics. Combined with their inherent trafficking ability and versatility of cargo capacity, exosomes can be engineered to specifically target cancerous cells, thereby minimizing off-target effects, and increasing the efficacy of cancer therapeutics. Exosomal formulations have mitigated the toxic effects of several drugs in murine cancer models. In this article, we review studies related to exosomal delivery of both small molecules and biologics, including siRNA to inhibit specific gene expression, in the pursuit of effective cancer therapeutics. We focus primarily on bovine milk and colostrum exosomes as the cancer therapeutic delivery vehicles based on their high abundance, cost effectiveness, scalability, high drug loading, functionalization of exosomes for targeted delivery, and lack of toxicity. While bovine milk exosomes may provide a new platform for drug delivery, extensive comparison to other nanoformulations and evaluation of long-term toxicity will be required to fully realize its potential.

Building Capability and Capacity: The Establishment of an Oncology Unit in the Solomon Islands.

PURPOSE: To describe the establishment of an oncology unit at the National Referral Hospital (NRH) in the Solomon Islands, a low-income nation in the South Pacific. METHODS: A scoping visit was carried out in 2016 to assist in the development of coordinated cancer services and to establish a medical oncology unit at the NRH at the request of the Medical Superintendent. This was followed by an observership visit to Canberra by an NRH doctor training in oncology in 2017. After a request from the Solomon Islands Ministry of Health, the Australian Government Department of Foreign Affairs and Trade (DFAT) arranged an in-country multidisciplinary mission under the Royal Australasian College of Surgeons/Royal Australasian College of Physicians Pacific Islands Program to help in the commissioning of the NRH Medical Oncology Unit in September 2018. Staff training and education sessions were held. The team, with the assistance of an Australian Volunteers International Pharmacist, has helped the NRH staff to develop localized Solomon Islands Oncology Guidelines. Donated equipment and supplies have helped with the initial establishment of the service. A second DFAT Oncology mission visit was made in 2019 followed by two NRH oncology nurses visiting Canberra on observership later that year and support of the Solomon's doctor to pursue postgraduate education in cancer sciences. Ongoing mentorship and support has been maintained. RESULTS: The island nation now has a sustainable oncology unit delivering chemotherapy treatments and management of patients with cancer. CONCLUSION: A collaborative multidisciplinary team approach by professionals from the high-income country working with colleagues from the low-income nation with coordination of different stakeholders was the key to this successful initiative in improving cancer care.

Oxidative DNA damage following microsome/Cu(II)-mediated activation of the estrogens, 17ß-estradiol, equilenin, and equilin: role of reactive oxygen species.

Experimental and epidemiological data associate the exposure of estrogens to cancer development in several tissues, particularly, the breast, endometrium, liver, and kidney. One plausible mechanism of estrogen-mediated carcinogenicity is DNA damage by redox cycling of estrogen catechols. Reports have shown that metabolism of estrogens results in 2- and 4-hydroxylation to catechol metabolites which can then redox cycle. We examined the capacity of the endogenous estrogen, 17ß-estradiol, and two equine estrogens which formulate a significant proportion of hormone replacement drugs, equilenin and equilin, to induce oxidatively generated DNA damage. Microsome/Cu(II)-mediated activation of all three estrogens resulted in numerous oxidation DNA adducts, as detected by (32)P-postlabeling/TLC. Essentially the same DNA oxidation pattern was also found when catechol estrogens were incubated with DNA in the presence of Cu(II) suggesting that redox cycling of catechol estrogens mediates the formation of these DNA adducts. Since the oxidation patterns induced by estrogen catechols and other chemically diverse catechols were chromatographically identical to those generated by Fenton-type chemistry and these adducts were inhibited by known ROS modifiers (up to 96%), this oxidatively generated DNA damage is believed to be the product of the attack of free radicals on DNA, rather than direct addition of the estrogen quinones. These data support a mechanistic role by endogenous and synthetic estrogens to induce oxidative DNA damage in addition to specific DNA adducts.

A model system for antiviral siRNA therapeutics using exosome-based delivery.

Emerging viral diseases, such as Ebola, SARS, MERS, and the pathogen causing COVID-19, SARS-CoV-2, present a challenge for the development of therapeutics because of strict biosafety handling procedures and rapid mutation of their genomes. To facilitate the development of an adaptable and testable therapeutic model system, a colostrum exosome-based nanoparticle delivery system, EPM (exosome-PEI matrix), that overcomes stringent biosafety containment, was used to mimic the expression of viral proteins. This system would greatly expand the number of laboratories actively participating in the screening of potential therapeutics. EPM technology can deliver both plasmid DNA and siRNA to both simulate viral gene expression and screen potential antiviral siRNA therapeutics. Using this nanoplatform, three key SARS-CoV-2 proteins (the spike glycoprotein, nucleocapsid, and replicase) were expressed in vitro and in vivo. In vitro, several viral gene-targeting siRNAs were screened to determine knockdown efficiency, with some siRNA duplexes resulting in 80%-95% knockdown of corresponding protein expression. Moreover, in vivo experiments introducing the spike protein and nucleocapsid by EPM resulted in the production of antibodies against the viral antigen, measured up to 45 d after target delivery. Together, these findings support the efficacy of the EPM delivery system to establish a model for screening antiviral therapeutics-reduced biosafety level.

Erratum: A model system for antiviral siRNA therapeutics using exosome-based delivery.

[This corrects the article DOI: 10.1016/j.omtn.2022.08.011.].

Sustained systemic delivery of green tea polyphenols by polymeric implants significantly diminishes benzo[a]pyrene-induced DNA adducts.

The polyphenolics in green tea are believed to be the bioactive components. However, poor bioavailability following ingestion limits their efficacy in vivo. In this study, polyphenon E (poly E), a standardized green tea extract, was administered by sustained-release polycaprolactone implants (two, 2-cm implants; 20% drug load) grafted subcutaneously or via drinking water (0.8% w/v) to female S/D rats. Animals were treated with continuous low dose of benzo[a]pyrene (BP) via subcutaneous polymeric implants (2 cm; 10% load) and euthanized after 1 and 4 weeks. Analysis of lung DNA by (32)P-postlabeling resulted in a statistically significant reduction (50%; p = 0.023) of BP-induced DNA adducts in the implant group; however, only a modest (34%) but statistically insignificant reduction occurred in the drinking water group at 1 week. The implant delivery system also showed significant reduction (35%; p = 0.044) of the known BP diolepoxide-derived DNA adduct after 4 weeks. Notably, the total dose of poly E administered was >100-fold lower in the implant group than the drinking water group (15.7 versus 1,632 mg, respectively). Analysis of selected phase I, phase II, and nucleotide excision repair enzymes at both mRNA and protein levels showed no significant modulation by poly E, suggesting that the reduction in the BP-induced DNA adducts occurred presumably due to known scavenging of the antidiolepoxide of BP by the poly E catechins. In conclusion, our study demonstrated that sustained systemic delivery of poly E significantly reduced BP-induced DNA adducts in spite of its poor bioavailability following oral administration.

Indigenous Community-Led Programs to Address Food and Water Security: Protocol for a Systematic Review.

The connection between indigenous peoples and Country (a multidimensional concept including land and water) enabled communities to thrive and survive over millennia. This has been eroded by colonisation, dispossession and increasing food and water insecurity due to climate change and supply constraints. Globally, indigenous peoples experience a disproportionate burden of chronic disease and poor nutrition is a major risk factor. Indigenous leaders have been advocating for community-led solutions. The primary aim of this systematic review is to determine what community-led programs have been undertaken to address food and/or water security globally. A comprehensive search of peer-reviewed literature will be performed in EMBASE, CINAHL, PsycINFO, PubMed, Scopus, LILACs, Informit and Business Source Premier. The grey literature search will include grey literature databases, customised Google search engines, targeted websites, and consultation with experts. The search strategy will consist of four concepts, combined as follows: (1) indigenous peoples AND (2) community program AND (3) food security OR (4) water security. Covidence will be used for study screening and data extraction by two authors. A deductive thematic analysis using indigenous-informed methodologies will be used to synthesise data. This review seeks to provide insight on models and mechanisms to encourage action and metrics for quantifying success of indigenous community-led programs to improve food and water security.

Targeted Oral Delivery of Paclitaxel Using Colostrum-Derived Exosomes.

Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small-cell lung cancer (NSCLC) is the most common type accounting for 84% of all lung cancers. Paclitaxel (PAC) is a widely used drug in the treatment of a broad spectrum of human cancers, including lung. While efficacious, PAC generally is not well tolerated and its limitations include low aqueous solubility, and significant toxicity. To overcome the dose-related toxicity of solvent-based PAC, we utilized bovine colostrum-derived exosomes as a delivery vehicle for PAC for the treatment of lung cancer. Colostrum provided higher yield of exosomes and could be loaded with higher amount of PAC compared to mature milk. Exosomal formulation of PAC (ExoPAC) showed higher antiproliferative activity and inhibition of colony formation against A549 cells compared with PAC alone, and also showed antiproliferative activity against a drug-resistant variant of A549. To further enhance its efficacy, exosomes were attached with a tumor-targeting ligand, folic acid (FA). FA-ExoPAC given orally showed significant inhibition (>50%) of subcutaneous tumor xenograft while similar doses of PAC showed insignificant inhibition. In the orthotopic lung cancer model, oral dosing of FA-ExoPAC achieved greater efficacy (55% growth inhibition) than traditional i.v. PAC (24-32% growth inhibition) and similar efficacy as i.v. Abraxane (59% growth inhibition). The FA-ExoPAC given i.v. exceeded the therapeutic efficacy of Abraxane (76% growth inhibition). Finally, wild-type animals treated with p.o. ExoPAC did not show gross, systemic or immunotoxicity. Solvent-based PAC caused immunotoxicity which was either reduced or completely mitigated by its exosomal formulations. These studies show that a tumor-targeted oral formulation of PAC (FA-ExoPAC) significantly improved the overall efficacy and safety profile while providing a user-friendly, cost-effective alternative to bolus i.v. PAC and i.v. Abraxane.

Exosome-mediated delivery of RNA and DNA for gene therapy.

Gene therapy promises to revolutionize biomedicine and personalized medicine by modulating or compensating the expression of abnormal genes. The biggest obstacle for clinical application is the lack of an effective, non-immunogenic delivery system. We show that bovine colostrum exosomes and polyethyleneimine matrix (EPM) delivers short interfering RNA (siRNA) or plasmid DNA (pDNA) for effective gene therapy. KRAS, a therapeutic focus for many cancers, was targeted by EPM-delivered KRAS siRNA (siKRAS) and inhibited lung tumor growth (>70%) and reduced KRAS expression (50%-80%). Aberrant p53 is another therapeutic focus for many cancers. EPM-mediated introduction of wild-type (WT) p53 pDNA (pcDNA-p53) resulted in p53 expression in p53-null H1299 cells in culture, subcutaneous lung tumor, and tissues of p53-knockout mice. Additionally, chemo-sensitizing effects of paclitaxel were restored by exogenous WT p53 in lung cancer cells. Together, this novel EPM technology represents an effective 'platform' for delivery of therapeutic nucleic acids to treat human disease.

Formation and differential repair of covalent DNA adducts generated by treatment of human cells with (+/-)-anti-dibenzo[a,l]pyrene-11,12-diol-13,14-epoxide.

Dibenzo[a,l]pyrene (DBP) is the most potent tumor initiating polycyclic aromatic hydrocarbon tested to date in rodent tumor models. To investigate how DBP adduct formation and removal might influence carcinogenesis, we have examined the effects of treatment of several nucleotide excision repair (NER)-proficient (NER(+)) and -deficient (NER(-)) cell lines with the carcinogenic metabolite (+/-)-anti-DBP-11,12-diol-13,14-epoxide (DBPDE). The treatment of NER(-) cells with (+/-)-anti-DBPDE for 0.5, 1, or 2 h yielded similar total adduct levels, indicating that adduct formation was essentially complete during a 2 h treatment period with no additional adducts produced after replacement of media. In all cell lines, treatment with (+/-)-anti-DBPDE generated five major and at least two minor adducts that were chromatographically identical to those formed by direct treatment of 3'-GMP and 3'-AMP with (+/-)-anti-DBPDE. When adduct levels were assessed in NER(-) cells, the number of adducts/10(9) nucleotides decreased over time, suggesting that DNA replication was ongoing, so we incorporated a normalization strategy based on DNA synthesis. This strategy indicated that DBPDE-DNA adduct levels in NER(-) cells are stable over time. After normalization for DNA synthesis in the NER(+) cells, our data indicated that three adducts showed biphasic repair kinetics. A faster rate of removal was observed during the first 6 h following DBPDE removal followed by a slower rate for up to 34 h. Importantly, two of the major guanine adducts were particularly refractory to removal in the NER(+) cells. Our results suggest that the extreme carcinogenicity of DBPDE may result from the ability of a substantial percentage of two structurally distinct DBPDE-DNA adducts to escape repair.

Oxidatively generated DNA damage after Cu(II) catalysis of dopamine and related catecholamine neurotransmitters and neurotoxins: Role of reactive oxygen species.

There is increasing evidence supporting a causal role for oxidatively damaged DNA in neurodegeneration during the natural aging process and in neurodegenerative diseases such as Parkinson and Alzheimer. The presence of redox-active catecholamine neurotransmitters coupled with the localization of catalytic copper to DNA suggests a plausible role for these agents in the induction of oxidatively generated DNA damage. In this study we have investigated the role of Cu(II)-catalyzed oxidation of several catecholamine neurotransmitters and related neurotoxins in inducing oxidatively generated DNA damage. Autoxidation of all catechol neurotransmitters and related congeners tested resulted in the formation of nearly a dozen oxidation DNA products resulting in a decomposition pattern that was essentially identical for all agents tested. The presence of Cu(II), and to a lesser extent Fe(III), had no effect on the decomposition pattern but substantially enhanced the DNA product levels by up to 75-fold, with dopamine producing the highest levels of unidentified oxidation DNA products (383±46 adducts/10(6) nucleotides), nearly 3-fold greater than 8-oxo-7,8-dihydro-2'-deoxyguanosine (122±19 adducts/10(6) nucleotides) under the same conditions. The addition of sodium azide, 2,2,6,6-tetramethyl-4-piperidone, tiron, catalase, bathocuproine, or methional to the dopamine/Cu(II) reaction mixture resulted in a substantial decrease (>90%) in oxidation DNA product levels, indicating a role for singlet oxygen, superoxide, H(2)O(2), Cu(I), and Cu(I)OOH in their formation. Whereas the addition of N-tert-butyl-α-phenylnitrone significantly decreased (67%) dopamine-mediated oxidatively damaged DNA, three other hydroxyl radical scavengers, ascorbic acid, sodium benzoate, and mannitol, had little to no effect on these oxidation DNA product levels, suggesting that free hydroxyl radicals may have limited involvement in this dopamine/Cu(II)-mediated oxidatively generated DNA damage. These studies suggest a possible contributory role of oxidatively generated DNA damage by dopamine and related catechol neurotransmitters/neurotoxins in neurodegeneration and cell death. We also found that a naturally occurring broad-spectrum antioxidant, ellagic acid, was substantially effective (nearly 50% inhibition) at low doses (1µM) at preventing this dopamine/Cu(II)-mediated oxidatively generated DNA damage. Because dietary ellagic acid has been found to reduce oxidative stress in rat brains, a neuroprotective role of this polyphenol is plausible.

Oxidative DNA adducts after Cu(2+)-mediated activation of dihydroxy PCBs: role of reactive oxygen species.

Polychlorinated biphenyls (PCBs) are toxic industrial chemicals, complete carcinogens, and efficacious tumor promoters. However, the mechanism(s) of PCB-mediated carcinogenicity remains largely undefined. One likely pathway by which these agents may play a role in carcinogenesis is the generation of oxidative DNA damage by redox cycling of dihydroxylated PCB metabolites. We have now employed a new (32)P-postlabeling system to examine novel oxidative DNA lesions induced by Cu(2+)-mediated activation of PCB metabolites. (32)P postlabeling of DNA incubated with various PCB metabolites resulted in over a dozen novel polar oxidative DNA adducts that were chromatographically similar for all active agents. The most potent metabolites tested were the hydroquinones (hydroxyl groups arranged para to each other), yielding polar oxidative adduct levels ranging from 55 to 142 adducts/10(6) nucleotides. PCB catechols, or ortho-dihydroxy metabolites, were up to 40% less active than their corresponding hydroquinone congeners, whereas monohydroxylated and quinone metabolites did not produce detectable oxidative damage over that of vehicle. With the exception of 2,4,5-Cl-2',5'-dihydroxybiphenyl, this oxidative DNA damage seemed to be inversely related to chlorine content: no chlorine approximately mono->di->trichlorinated metabolites. Importantly, copper, but not iron, was essential for activation of the PCB metabolites to these polar oxidative DNA adducts, because in its absence or in the presence of the Cu(+)-specific scavenger bathocuproine, no adducts were detected. Intervention studies with known reactive oxygen species (ROS) modifiers suggested that H(2)O(2), singlet oxygen, hydroxyl radical, and superoxide may also be involved in this PCB-mediated oxidative DNA damage. These data indicate a mechanistic role for several ROS, in addition to copper, in PCB-induced DNA damage and provide further support for oxidative DNA damage in PCB-mediated carcinogenesis.