Unraveling Links between Chronic Inflammation and Long COVID: Workshop Report.

As COVID-19 continues, an increasing number of patients develop long COVID symptoms varying in severity that last for weeks, months, or longer. Symptoms commonly include lingering loss of smell and taste, hearing loss, extreme fatigue, and "brain fog." Still, persistent cardiovascular and respiratory problems, muscle weakness, and neurologic issues have also been documented. A major problem is the lack of clear guidelines for diagnosing long COVID. Although some studies suggest that long COVID is due to prolonged inflammation after SARS-CoV-2 infection, the underlying mechanisms remain unclear. The broad range of COVID-19's bodily effects and responses after initial viral infection are also poorly understood. This workshop brought together multidisciplinary experts to showcase and discuss the latest research on long COVID and chronic inflammation that might be associated with the persistent sequelae following COVID-19 infection.

Extracellular vesicles: potential applications in cancer diagnosis, prognosis, and epidemiology.

Both normal and diseased cells continuously shed extracellular vesicles (EVs) into extracellular space, and the EVs carry molecular signatures and effectors of both health and disease. EVs reflect dynamic changes that are occurring in cells and tissue microenvironment in health and at a different stage of a disease. EVs are capable of altering the function of the recipient cells. Trafficking and reciprocal exchange of molecular information by EVs among different organs and cell types have been shown to contribute to horizontal cellular transformation, cellular reprogramming, functional alterations, and metastasis. EV contents may include tumor suppressors, phosphoproteins, proteases, growth factors, bioactive lipids, mutant oncoproteins, oncogenic transcripts, microRNAs, and DNA sequences. Therefore, the EVs present in biofluids offer unprecedented, remote, and non-invasive access to crucial molecular information about the health status of cells, including their driver mutations, classifiers, molecular subtypes, therapeutic targets, and biomarkers of drug resistance. In addition, EVs may offer a non-invasive means to assess cancer initiation, progression, risk, survival, and treatment outcomes. The goal of this review is to highlight the current status of information on the role of EVs in cancer, and to explore the utility of EVs for cancer diagnosis, prognosis, and epidemiology.

Correlation between CYP1A1 transcript, protein level, enzyme activity and DNA adduct formation in normal human mammary epithelial cell strains exposed to benzo[a]pyrene.

The polycyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (BP) is thought to bind covalently to DNA, through metabolism by cytochrome P450 1A1 (CYP1A1) and CYP1B1, and other enzymes, to form r7, t8, t9-trihydroxy-c-10-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]-pyrene (BPdG). Evaluation of RNA expression data, to understand the contribution of different metabolic enzymes to BPdG formation, is typically presented as fold-change observed upon BP exposure, leaving the actual number of RNA transcripts unknown. Here, we have quantified RNA copies/ng cDNA (RNA cpn) for CYP1A1 and CYP1B1, as well as NAD(P)H: quinone oxidoreductase 1 (NQO1), which may reduce formation of BPdG adducts, using primary normal human mammary epithelial cell (NHMEC) strains, and the MCF-7 breast cancer cell line. In unexposed NHMECs, basal RNA cpn values were 58-836 for CYP1A1, 336-5587 for CYP1B1 and 5943-40112 for NQO1. In cells exposed to 4.0 µM BP for 12h, RNA cpn values were 251-13234 for CYP1A1, 4133-57078 for CYP1B1 and 4456-55887 for NQO1. There were 3.5 (mean, range 0.2-15.8) BPdG adducts/10(8) nucleotides in the NHMECs (n = 16), and 790 in the MCF-7s. In the NHMECs, BP-induced CYP1A1 RNA cpn was highly associated with BPdG (P = 0.002), but CYP1B1 and NQO1 were not. Western blots of four NHMEC strains, chosen for different levels of BPdG adducts, showed a linear correlation between BPdG and CYP1A1, but not CYP1B1 or NQO1. Ethoxyresorufin-O-deethylase (EROD) activity, which measures CYP1A1 and CYP1B1 together, correlated with BPdG, but NQO1 activity did not. Despite more numerous levels of CYP1B1 and NQO1 RNA cpn in unexposed and BP-exposed NHMECs and MCF-7cells, BPdG formation was only correlated with induction of CYP1A1 RNA cpn. The higher level of BPdG in MCF-7 cells, compared to NHMECs, may have been due to a much increased induction of CYP1A1 and EROD. Overall, BPdG correlation was observed with CYP1A1 protein and CYP1A1/1B1 enzyme activity, but not with CYP1B1 or NQO1 protein, or NQO1 enzyme activity.

Perinatal exposure of patas monkeys to antiretroviral nucleoside reverse-transcriptase inhibitors induces genotoxicity persistent for up to 3 years of age.

BACKGROUND: Erythrocebus patas (patas) monkeys were used to model antiretroviral (ARV) drug in human immunodeficiency virus type 1-infected pregnant women. METHODS: Pregnant patas dams were given human-equivalent doses of ARVs daily during 50% of gestation. Mesenchymal cells, cultured from bone marrow of patas offspring obtained at birth and at 1 and 3 years of age, were examined for genotoxicity, including centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes. RESULTS: Compared with controls, statistically significant increases (P < .05) in centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes were found in mesenchymal cells from most groups of offspring at the 3 time points. CONCLUSIONS: Transplacental nucleoside reverse-transcriptase inhibitor exposures induced fetal genotoxicity that was persistent for 3 years.

Next generation analytic tools for large scale genetic epidemiology studies of complex diseases.

Over the past several years, genome-wide association studies (GWAS) have succeeded in identifying hundreds of genetic markers associated with common diseases. However, most of these markers confer relatively small increments of risk and explain only a small proportion of familial clustering. To identify obstacles to future progress in genetic epidemiology research and provide recommendations to NIH for overcoming these barriers, the National Cancer Institute sponsored a workshop entitled "Next Generation Analytic Tools for Large-Scale Genetic Epidemiology Studies of Complex Diseases" on September 15-16, 2010. The goal of the workshop was to facilitate discussions on (1) statistical strategies and methods to efficiently identify genetic and environmental factors contributing to the risk of complex disease; and (2) how to develop, apply, and evaluate these strategies for the design, analysis, and interpretation of large-scale complex disease association studies in order to guide NIH in setting the future agenda in this area of research. The workshop was organized as a series of short presentations covering scientific (gene-gene and gene-environment interaction, complex phenotypes, and rare variants and next generation sequencing) and methodological (simulation modeling and computational resources and data management) topic areas. Specific needs to advance the field were identified during each session and are summarized.

The resveratrol analogue, 2,3',4,5'-tetramethoxystilbene, does not inhibit CYP gene expression, enzyme activity and benzo[a]pyrene-DNA adduct formation in MCF-7 cells exposed to benzo[a]pyrene.

Exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs) induces cytochrome P450 (CYP) 1A1 and 1B1 enzymes, which biotransform PAHs resulting in the formation of DNA adducts. We hypothesised that 2,3',4,5'-tetramethoxystilbene (TMS), an analogue of resveratrol and a potent CYP1B1 inhibitor, may inhibit r7, t8, t9-trihydroxy-c-10-(N(2)deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) adduct formation in cells exposed to benzo[a]pyrene (BP). To address this, MCF-7 cells were cultured for 96 h in the presence of 1 µM BP, 1 µM BP + 1 µM TMS or 1 µM BP + 4 µM TMS. Cells were assayed at 2-12 h intervals for: BPdG adducts by r7, t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence immunoassay; CYP1A1 and 1B1 gene expression changes by relative real-time polymerase chain reaction; and CYP1A1/1B1 enzyme activity by ethoxyresorufin-O-deethylase (EROD) assay. Whereas maximal BPdG levels were similar for all exposure groups, the times at which the maxima were reached increased by 16 and 24 h with the addition of 1 and 4 µM TMS, respectively. The maximal expression of CYP1A1 and CYP1B1 occurred at 16, 24 and 48 h, but the maximal level for EROD-specific activity was reached at 24, 48 and 60 h, in cells exposed to 1 µM BP, 1 µM BP + 1 µM TMS or 1 µM BP + 4 µM TMS, respectively. The area under the curve from 4 to 96 h of exposure (AUC(4-)(96 h)) for BPdG adduct formation was not increased in the presence of TMS, but for CYP1A1 and CYP1B1 expression fold increase AUC(4-)(96 h) and EROD-specific activity AUC(4-)(96 h), there were significant (P < 0.05) increases in the presence of 4 µM TMS. Therefore, during 96 h of exposure in MCF-7 cells, the combination of BP plus TMS caused a slowing of BP biotransformation, with an increase in CYP1A1 and CYP1B1 expression and EROD activity, and a slowing, but no change in magnitude of BPdG formation.

Epidemiologic Research of Rare Cancers: Trends, Resources, and Challenges.

BACKGROUND: The goals of this project were to assess the status of NCI's rare cancer-focused population science research managed by the Division of Cancer Control and Population Sciences (DCCPS), to develop a framework for evaluation of rare cancer research activities, and to review available resources to study rare cancers. METHODS: Cancer types with an overall age-adjusted incidence rate of less than 20 cases per 100,000 individuals were identified using NCI Surveillance, Epidemiology and End Results (SEER) Program data. SEER data were utilized to develop a framework based on statistical commonalities. A portfolio analysis of DCCPS-supported active grants and a review of three genomic databases were conducted. RESULTS: For the 45 rare cancer types included in the analysis, 123 active DCCPS-supported rare cancer-focused grants were identified, of which the highest percentage (18.7%) focused on ovarian cancer. The developed framework revealed five clusters of rare cancer types. The cluster with the highest number of grants (n = 43) and grants per cancer type (10.8) was the cluster that included cancer types of higher incidence, average to better survival, and high prevalence (in comparison with other rare cancers). Resource review revealed rare cancers are represented in available genomic resources, but to a lesser extent compared with more common cancers. CONCLUSIONS: This article provides an overview of the rare cancer-focused population sciences research landscape as well as information on gaps and opportunities. IMPACT: The findings of this article can be used to develop efficient and comprehensive strategies to accelerate rare cancer research.See related commentary by James V. Lacey Jr, p. 1300.

Polycyclic Aromatic Hydrocarbon-DNA Adducts in Gulf of Mexico Sperm Whale Skin Biopsies Collected in 2012.

The northern Gulf of Mexico has a long history of polycyclic aromatic hydrocarbon (PAH) contamination from anthropogenic activities, natural oil seepages, and the 2010 Deepwater Horizon explosion and oil spill. The continental shelf of the same area is a known breeding ground for sperm whales (Physeter macrocephalus). To evaluate PAH-DNA damage, a biomarker for potential cancer risk, we compared skin biopsies collected from Gulf of Mexico sperm whales in 2012 with skin biopsies collected from sperm whales in areas of the Pacific Ocean in 1999-2001. All samples were obtained by crossbow and comprised both epidermis and subcutaneous blubber. To evaluate exposure, 7 carcinogenic PAHs were analyzed in lipids extracted from Pacific Ocean sperm whale blubber, pooled by sex, and location. To evaluate PAH-DNA damage, portions of all tissue samples were formalin-fixed, paraffin-embedded, sectioned, and examined for PAH-DNA adducts by immunohistochemistry (IHC) using an antiserum elicited against benzo[a]pyrene-modified DNA, which crossreacts with several high molecular weight carcinogenic PAHs bound to DNA. The IHC showed widespread epidermal nuclear localization of PAH-DNA adducts in the Gulf of Mexico whales (n = 15) but not in the Pacific Ocean whales (n = 4). A standard semiquantitative scoring system revealed significantly higher PAH-DNA adducts in the Gulf of Mexico whales compared to the whales from the Pacific Ocean study (p = .0002).

In vivo localization and postmortem stability of benzo[a]pyrene-DNA adducts.

DNA adducts of carcinogenic polycyclic aromatic hydrocarbons (PAHs) play a critical role in the etiology of gastrointestinal tract cancers in humans and other species orally exposed to PAHs. Yet, the precise localization of PAH-DNA adducts in the gastrointestinal tract, and the long-term postmortem PAH-DNA adduct stability are unknown. To address these issues, the following experiment was performed. Mice were injected intraperitoneally with the PAH carcinogen benzo[a]pyrene (BP) and euthanized at 24 h. Tissues were harvested either at euthanasia (0 time), or after 4, 8, 12, 24, 48, and 168 hr (7 days) of storage at 4°C. Portions of mouse tissues were formalin-fixed, paraffin-embedded, and immunohistochemically (IHC) evaluated by incubation with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA antiserum and H-scoring. The remaining tissues were frozen, and DNA was extracted and assayed for the r7,t8,t9-trihydroxy-c-10-(N 2 -deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG) adduct using two quantitative assays, the BPDE-DNA chemiluminescence immunoassay (CIA), and high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ES-MS/MS). By IHC, which required intact nuclei, BPdG adducts were visualized in forestomach basal cells, which included gastric stem cells, for up to 7 days. In proximal small intestine villus epithelium BPdG adducts were visualized for up to 12 hr. By BPDE-DNA CIA and HPLC-ES-MS/MS, both of which used DNA for analysis and correlated well (P= 0.0001), BPdG adducts were unchanged in small intestine, forestomach, and lung stored at 4°C for up to 7 days postmortem. In addition to localization of BPdG adducts, this study reveals the feasibility of examining PAH-DNA adduct formation in wildlife species living in colder climates. Environ. Mol. Mutagen. 61:216-223, 2020. © 2019 Wiley Periodicals, Inc.

Circulating Tumor DNA Assays in Clinical Cancer Research.

The importance of circulating free DNA (cfDNA) in cancer clinical research was recognized in 1994 when a mutated RAS gene fragment was detected in a patient's blood sample. Up to 1% of the total circulating DNA in patients with cancer is circulating tumor DNA (ctDNA) that originates from tumor cells. As ctDNA is rapidly cleared from the blood stream and can be obtained by minimally invasive methods, it can be used as a dynamic cancer biomarker for cancer early detection, diagnosis, and treatment monitoring. Despite the potential for clinical use, few ctDNA assays have been cleared or approved by the US Food and Drug Administration. As tools for clinical and translational research, current ctDNA assays face some challenges, and more research is needed to advance use of these assays. On September 29-30, 2016, the Division of Cancer Treatment and Diagnosis at the National Cancer Institute convened a workshop entitled "Circulating Tumor DNA Assays in Clinical Cancer Research" to garner input from industry experts, academia, and government research and regulatory agencies to understand and promote the translation of ctDNA assays to clinical research, with potential to advance to use in clinical practice. This Commentary presents the topics of the workshop covered in the presentations and points made in the discussions that followed: 1) background on ctDNA, 2) potential clinical utility of ctDNA assays, 3) assay technology, 4) assay clinical and analytical validation, and 5) industry perspectives. Additional relevant information that has come to light since the workshop has been included.

Erythrocebus patas monkey offspring exposed perinatally to NRTIs sustain skeletal muscle mitochondrial compromise at birth and at 1 year of age.

Antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs), given to human immunodeficiency virus-1-infected pregnant women to prevent vertical viral transmission, have caused mitochondrial dysfunction in some human infants. Here, we examined mitochondrial integrity in skeletal muscle from offspring of pregnant retroviral-free Erythrocebus patas dams administered human-equivalent NRTI doses for the last 10 weeks of gestation or for 10 weeks of gestation and 6 weeks after birth. Exposures included no drug, Zidovudine (AZT), Lamivudine (3TC), AZT/3TC, AZT/Didanosine (ddI), and Stavudine (d4T)/3TC. Offspring were examined at birth (n=3 per group) and 1 year (n=4 per group, not including 3TC alone). Circulating levels of creatine kinase were elevated at 1 year in the d4T/3TC-exposed group. Measurement of oxidative phosphorylation enzyme activities (complexes I, II, and IV) revealed minimal NRTI-induced changes at birth and at 1 year. Histochemistry for complex IV activity showed abnormal staining with activity depletion at birth and 1 year in groups exposed to AZT alone and to the 2-NRTI combinations. Electron microscopy of skeletal muscle at birth and 1 year of age showed mild to severe mitochondrial damage in all the NRTI-exposed groups, with 3TC inducing mild damage and the 2-NRTI combinations inducing extensive damage. At birth, mitochondrial DNA (mtDNA) was depleted by approximately 50% in groups exposed to AZT alone and the 2-NRTI combinations. At 1 year, the mtDNA levels had increased but remained significantly below normal. Therefore, skeletal muscle mitochondrial compromise occurs at birth and persists at 1 year of age (46 weeks after the last NRTI exposure) in perinatally exposed young monkeys, suggesting that similar events may occur in NRTI-exposed human infants.

Transplacentally exposed human and monkey newborn infants show similar evidence of nucleoside reverse transcriptase inhibitor-induced mitochondrial toxicity.

Effective reduction in maternal-fetal human immunodeficiency virus-1 (HIV-1) transmission has been achieved by administration of nucleoside reverse transcriptase inhibitors (NRTIs) during pregnancy, and although most exposed children are clinically normal at birth, mitochondrial dysfunction has been reported. To examine mitochondrial integrity on a molecular level, we evaluated mitochondrial morphology by electron microscopy (EM) and mitochondrial DNA (mtDNA) quantity in umbilical cords and cord blood from NRTI-exposed and unexposed human and monkey newborns. Human subjects included infants born to HIV-1-infected mothers who received Combivir (Zidovudine [AZT] plus Lamivudine [3TC]) (n = 9) or AZT plus Didanosine [ddI] (n = 2) during pregnancy, and infants born to HIV-1-uninfected mothers (n = 7). NRTI-exposed Erythrocebus patas monkey dams (n = 3 per treatment group) were given human-equivalent dosing regimens containing 3TC, AZT/3TC, AZT/ddI, or Stavudine (d4T)/3TC during gestation. Four infants born to unexposed patas dams served as controls. Mitochondria in umbilical cord endothelial cells from NRTI-exposed monkey and human infants showed substantial abnormal pathology by EM, the extent of which was quantified from coded photomicrographs and shown to be different (P < 0.05) from the unexposed monkey and human newborns. Significant (P < 0.05) mtDNA depletion was found in umbilical cords from both human and monkey NRTI-exposed infants and in human, but not in monkey, cord blood leukocytes. For umbilical cords, an increase in mitochondrial morphological damage correlated with reduction in mtDNA quantity in fetal monkeys (r = 0.94). The treatment-induced mitochondrial compromise in infant monkeys ranked as follows: d4T/3TC > AZT/ddI > AZT/3TC > 3TC. The study demonstrates that transplacental NRTI exposures induce similar mitochondrial damage in cord blood and umbilical cords taken from retroviral-uninfected monkey infants and from human infants born to HIV-1-infected women.

Morphological and molecular course of mitochondrial pathology in cultured human cells exposed long-term to Zidovudine.

Long-term use of antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs) as therapy for human immunodeficiency virus-1 (HIV-1) infection is limited by mitochondrial toxicity. Here we document mitochondrial pathology during the long-term culture of human HeLa cells in the presence or absence of the NRTI Zidovudine(R) (AZT, 800 muM) for up to 77-passages (p), with samples taken at early (p5-p11), middle (p36 and p37), and late (p70-p77) passages. Samples were analyzed for changes in mitochondrial morphology, mitochondrial (mt)DNA quantity, nuclear and mitochondrial gene expression, and mitochondrial membrane potential. Mitochondria showed abnormal proliferation at p5 and abnormal morphology >/=p36. mtDNA quantity was increased at p5 and p11, and 65% depleted at p71. Hierarchical clustering of nuclear gene expression, examined at p37 by the NCI cDNA microarray in AZT-exposed cells, showed down-regulation of 13 out of 16 lipid-metabolizing genes, and up-regulation of most oxidative phosphorylation (OXPHOS) genes. OXPHOS genes encoded by mtDNA, examined at p5, p36, and p75 using the Mitochondrial Gene Mini Array, revealed up-regulation of genes coding for polypeptides of NADH dehydrogenase, ATP synthase, and cytochrome c oxidase. Mitochondrial membrane potential, monitored by JC1 staining, was elevated at p10 and p32, and essentially completely absent at p71. The data show that during chronic exposure of HeLa cells to AZT, a compensatory response was induced at the earlier passages (p5-p37), and by p71 there was widespread mitochondrial morphological damage, severe mtDNA depletion, and a substantial loss of mitochondrial membrane potential.

Clinical Utility of Epstein-Barr Virus DNA Testing in the Treatment of Nasopharyngeal Carcinoma Patients.

Epstein-Barr virus (EBV) DNA analysis has been shown to be useful for early detection, prognostication, and monitoring of treatment response of nasopharyngeal carcinoma (NPC), and the recent literature provides growing evidence of the clinical utility of EBV DNA testing, particularly to inform treatment decisions for NPC patients. Despite the fact that NPC is a rare disease, the NRG Oncology cooperative group has successfully activated a phase 2/3 randomized clinical trial for NPC with international partners and in that process has discovered that the development of a harmonized EBV DNA test is absolutely critical for integration into clinical trials and for future deployment in clinical and central laboratories. In November 2015, the National Cancer Institute (NCI) convened a workshop of international experts in the treatment of NPC and EBV testing to provide a forum for discussing the state of EBV DNA testing and its clinical utility, and to stimulate consideration of future studies and clinical practice guidelines for EBV DNA. This review provides a summary of that discussion.

Formation of tamoxifen-DNA adducts in multiple organs of adult female cynomolgus monkeys dosed with tamoxifen for 30 days.

The use of the antiestrogen tamoxifen (TAM) is associated with an increase in endometrial cancer. TAM-induced endometrial carcinogenesis may proceed through a genotoxin-mediated pathway, although the detection of endometrial TAM-DNA adducts in exposed women is still controversial. In this study, a monkey model has been used to investigate the question of TAM-DNA adduct formation in primates. Two methods have been used to determine TAM-DNA adducts: a TAM-DNA chemiluminescence immunoassay (TAM-DNA CIA), using an antiserum that has specificity for (E)-alpha-(deoxyguanosin-N(2)-yl)-tamoxifen (dG-TAM) and (E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen (dG-desmethyl-TAM) and electrospray ionization tandem mass spectrometry (ES-MS/MS) coupled with on-line sample preparation and high-performance liquid chromatography (HPLC). Mature (19 year old) cynomolgus monkeys were given either vehicle control (n = 1) or TAM (n = 3) twice daily for a total dose of 2 mg of TAM/kg body weight (bw)/day for 30 days by naso-gastric intubation. Tissues were harvested, and DNA was isolated from uterus, ovary, liver, brain cortex, and kidney. By TAM-DNA CIA, values for uterine TAM-DNA adducts in two monkeys were 0.9 and 1.7 adducts/10(8) nucleotides, whereas values for ovarian TAM-DNA adducts in the same animals were 0.4 and 0.5 adducts/10(8) nucleotides. Liver, brain cortex, and kidney DNA samples from the three exposed monkeys had TAM-DNA levels of 2.1-4.2 adducts/10(8) nucleotides, 0.4-5.0 adducts/10(8) nucleotides, and 0.7-2.1 adducts/10(8) nucleotides, respectively. By HPLC-ES-MS/MS, the levels of TAM-DNA adducts detected in all tissues were comparable with those observed by TAM-DNA CIA. Thus, values for uterine TAM-DNA adducts ranged from 0.5 to 1.4 adducts/10(8) nucleotides, whereas values for ovarian TAM-DNA adducts, measurable in two monkeys, were 0.2 and 0.3 adducts/10(8) nucleotides. Liver DNA contained the highest TAM-DNA adduct levels (7.0-11.1 adducts/10(8) nucleotides), whereas brain cortex DNA contained lower adduct levels (0.6-4.8 adducts/10(8) nucleotides) and the lowest levels were measured in the kidney (0.2-0.4 adducts/10(8) nucleotides). This study indicates that cynomolgus monkeys are capable of metabolizing TAM to genotoxic intermediates that form TAM-DNA adducts in multiple tissues.

Identification of tamoxifen-DNA adducts in monkeys treated with tamoxifen.

The risk of developing endometrial cancer is increased in breast cancer patients treated with tamoxifen (TAM) and in healthy women undergoing TAM chemoprevention. We have detected previously TAM-DNA adducts in the endometrium of women receiving TAM (Shibutani et al., Carcinogenesis, 21: 1461-1467, 2000). To investigate the genotoxic damage induced by TAM in the uterus and other tissues of primates, we gave adult female cynomolgus monkeys six times the human-equivalent dose of TAM (2 mg/kg body weight/day) for 30 days. DNA samples were prepared from the uterus, ovary, liver, kidney, and brain cortex of three TAM-exposed monkeys and one control monkey and were analyzed as coded specimens. To identify the TAM-DNA adducts, we established a new high-performance liquid chromatography gradient system for (32)P-postlabeling/high-performance liquid chromatography analysis, which can resolve the trans- and cis-diastereoisomers of alpha-(N(2)-deoxyguanosinyl)TAM (dG-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethylTAM, and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide. Trans-forms of dG-N(2)-TAM and dG-N(2)-N-desTAM adducts were detected in the livers of all three TAM-fed monkeys at levels of 2.7 adducts/10(8) nucleotides and 1.7 adducts/10(8) nucleotides, respectively. The levels of dG-N(2)-TAM adducts observed in the uterus of one monkey and in the ovaries of two monkeys were approximately 10-fold lower than those observed in the livers. TAM exposure also induced dG-N(2)-TAM adduct in the brain cortex of all three monkeys with a value of 1.5 adducts/10(8) nucleotides. No TAM-DNA adducts were detected in the kidneys or in any tissues obtained from the unexposed monkey. Our results suggest that women receiving TAM may form genotoxic damage in many organs, including the reproductive organs.

Mitochondrial compromise in 3-year old patas monkeys exposed in utero to human-equivalent antiretroviral therapies.

Antiretroviral (ARV) drug therapy, given during pregnancy for prevention of mother-to-child transmission of human immunodeficiency virus 1 (HIV-1), induces fetal mitochondrial dysfunction in some children. However, the persistence/reversibility of that dysfunction is unclear. Here we have followed Erythrocebus patas (patas) monkey offspring for up to 3 years of age (similar in development to a 15-year old human) after exposure of the dams to human-equivalent in utero ARV exposure protocols. Pregnant patas dams (3-5/exposure group) were given ARV drug combinations that included zidovudine (AZT)/lamivudine (3TC)/abacavir (ABC), or AZT/3TC/nevirapine (NVP), for the last 10 weeks (50%) of gestation. Infants kept for 1 and 3 years also received drug for the first 6 weeks of life. In offpsring at birth, 1 and 3 years of age mitochondrial morphology, examined by electron microscopy (EM), was compromised compared to the unexposed controls. Mitochondrial DNA (mtDNA), measured by hybrid capture chemiluminescence assay (HCCA) was depleted in hearts of patas exposed to AZT/3TC/NVP at all ages (P < 0.05), but not in those exposed to AZT/3TC/ABC at any age. Compared to unexposed controls, mitochondrial reserve capacity oxygen consumption rate (OCR by Seahorse) in cultured bone marrow mesenchymal fibroblasts from 3-year-old patas offspring was ∼50% reduced in AZT/3TC/ABC-exposed patas (P < 0.01), but not in AZT/3TC/NVP-exposed patas. Overall the data show that 3-year-old patas sustain persistent mitochondrial dysfunction as a result of perinatal ARV drug exposure. Environ. Mol. Mutagen. 57:526-534, 2016. © 2016 Wiley Periodicals, Inc.

Induction of CYP1A1 and CYP1B1 and formation of carcinogen-DNA adducts in normal human mammary epithelial cells treated with benzo[a]pyrene.

Inter-individual variation in formation of carcinogen-DNA adducts and induction of cytochrome P450 genes was measured in 23 cultured normal human mammary epithelial cell (NHMEC) strains established from reduction mammoplasty tissue. Semi-confluent cells were exposed to 4 microM benzo[a]pyrene (BP) for 12 h and BP-DNA adduct levels were measured by chemiluminescence immunoassay using antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). BP-DNA adduct levels for 22 of 23 different cell strains ranged from non-detectable (three samples) to about 15 adducts/10(8) nucleotides. Increases in levels of CYP1A1 and CYP1B1 were detected using both oligonucleotide arrays and reverse transcription/quantitative real-time polymerase chain reactions (RT-PCRs). For CYP1A1 and CYP1B1, the oligonucleotide array data and RT-PCR data were highly correlated (r=0.73 and 0.70, respectively), suggesting that oligonucleotide arrays are a suitable gene discovery tool, and demonstrating that the complementary and efficient RT-PCR may be used to confirm microarray data for a specific gene in a large number of samples. As measured by RT-PCR, inter-individual variation in CYP1A1 induction was 100-fold, while the variation in CYP1B1 induction was almost 40-fold. On a per-person basis, CYP1A1 and CYP1B1 induction were well-correlated (r=0.88, P<0.001), which is to be expected as they are under the control of a common transcriptional regulation mechanism in response to BP exposure. Inter-individual variation in carcinogen-DNA adduct formation could not be explained only by variation in levels of CYP1A1 or CYP1B1 induction, as neither was well-correlated with BPDE-DNA adduct level (r=0.40 and 0.50 for CYP1A1 and CYP1B1, respectively). Evaluation of glutathione-S-transferase M1 genotype (GSTM1 positive or null) revealed an apparent correlation between positive GSTM1 genotype and BPDE-DNA adduct levels (r=0.84 and 0.77 for CYP1A1 and CYP1B1, respectively); however, after removal of the single outlier this relationship was not significant. Overall the data suggest that BPDE-DNA adduct levels in normal human breast tissue may be modulated by multiple factors that include, but are not exclusive to, CYP1A1 and CYP1B1 inducibility and the presence or absence of GSTM1.

Cardiac mitochondrial compromise in 1-yr-old Erythrocebus patas monkeys perinatally-exposed to nucleoside reverse transcriptase inhibitors.

Hearts from 1-yr-old Erythrocebus patas monkeys were examined after in utero and 6-wk-postbirth exposure to antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs). Protocols were modeled on those given to human immunodeficiency virus (HIV)-1-infected pregnant women. NRTIs were administered daily to the dams for the last 20% or 50% of gestation, and to the infants for 6 wk after birth. Exposures included: no drug (n = 4); Zidovudine, 3'-azido-3'-deoxythymidine (AZT; n = 4); AZT/Lamivudine, (-)-beta-L-2', 3'-Dideoxy-3'-thiacytidine (Epivir, 3TC) (n = 4); AZT/Didanosine (Videx, ddI) (n = 4); and Stavudine (Zerit, d4T)/3TC (n = 4). Echocardiograms and clinical chemistry showed no drug-related changes, but the d4T/3TC-exposed fetuses at 6 and 12 mo had increased white cell counts (p < 0.05). At 1 yr of age, oxidative phosphorylation (OXPHOS) enzyme activities were similar in heart mitochondria from all groups. Mitochondrial pathology, that included clones of damaged mitochondria (p < 0.05), was found in hearts of all 1-yr drug-exposed infants. Levels of mtDNA were elevated (p < 0.05) in hearts of all NRTI-exposed monkeys in the following order: control < d4T/3TC < AZT < AZT/3TC < AZT/ddI. The clinical status of NRTI-exposed infants, as evidenced by behavior, clinical chemistry, OXPHOS activity and echocardiogram, was normal. However, extensive mitochondrial damage with clusters of similar-appearing damaged heart mitochondria observed by electron microscopy, and an increase in mtDNA quantity, that persisted at 1 yr of age, suggest the potential for cardiotoxicity later in life.