Higher Adduct Levels Research Articles

Electrophilic reactive aldehydes as a therapeutic target in colorectal cancer prevention and treatment.

Colorectal cancer (CRC) is a major health problem worldwide. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation and form covalent adducts with amine-containing macromolecules. We have shown high levels of adducts of isoLGs in colonic epithelial cells of patients with CRC. We thus investigated the role of these reactive aldehydes in colorectal cancer development. We found that 2-hydroxybenzylamine (2-HOBA), a natural compound derived from buckwheat seeds that acts as a potent scavenger of electrophiles, is bioavailable in the colon of mice after supplementation in the drinking water and does not affect the colonic microbiome. 2-HOBA reduced the level of isoLG adducts to lysine as well as tumorigenesis in models of colitis-associated carcinogenesis and of sporadic CRC driven by specific deletion of the adenomatous polyposis coli gene in colonic epithelial cells. In parallel, we found that oncogenic NRF2 activation and signaling were decreased in the colon of 2-HOBA-treated mice. Additionally, the growth of xenografted human HCT116 CRC cells in nude mice was significantly attenuated by 2-HOBA supplementation. In conclusion, 2-HOBA represents a promising natural compound for the prevention and treatment of CRC.

Interaction of polycyclic aromatic hydrocarbon DNA adducts and telomere length on missed abortion

Objective: To analyze the contribution and interaction of polycyclic aromatic hydrocarbons (PAH)-DNA adducts and changes of telomere length (TL) on missed abortion. Methods: From March to December 2019, patients with missed abortion in the First Hospital of Shanxi Medical University and pregnant women with normal pregnancy but voluntary abortion in the same department during the same period were selected and divided into a case group and a control group. Questionnaire was used to investigate the general situation and the pregnancy situation of the subjects. The abortion villi were collected and the content of PAH-DNA adducts and TL was detected. Logistic regression model was used to analyze the associated factors of missed abortion. R epiR package and Mediation package were used to analyze the effect and relationship between PAH-DNA adducts and TL on missed abortion. Results: The age of the subjects was(29.92±5.69)years old. The M(Q1,Q3)of PAH-DNA adducts was 453.75(404.61, 504.72) pg/ml. The M(Q1,Q3)of TL was 1.21(0.77, 1.72). The content of PAH-DNA adducts in the case group was higher than that in the control group (Z=-2.10, P=0.036), while the TL was lower than that in the control group (Z=-4.05, P<0.001). Multivariate logistic regression showed that low, medium and high levels of PAH-DNA adducts (OR=3.17,95%CI:1.41-7.14;OR=2.85,95%CI:1.25-6.52;OR=2.46,95%CI:1.07-5.64), and long, medium and short levels of TL (OR=2.50,95%CI:1.11-5.63;OR=3.32,95%CI:1.45-7.56;OR=3.22,95%CI:1.42-7.26) were all risk factors for missed abortion. The medium level of PAH-DNA adducts had a 2.76-fold higher risk of shortened TL than those with the lowest level, and no mediating role of TL was found. The stratified analysis showed that when the TL level was longer (>1.21), the low and high levels of PAH-DNA adducts were associated with missed abortion (all P<0.05); when the TL level was shorter (<1.21), the medium level of PAH-DNA adducts was associated with abortion (P=0.025). At lower levels of PAH-DNA adducts, no effect of TL on missed abortion was observed, while, at higher levels, TL was strongly associated with missed abortion (OR=7.50,95%CI:1.95-28.82;OR=6.04,95%CI:1.54-23.65;OR=9.05,95%CI:2.34-35.04). The interaction analysis found that the AP was 0.72 (95%CI: 0.46-0.99), and the SI was 5.21 (95%CI: 2.30-11.77). Conclusion: The high level of PAH-DNA adducts and shortened TL may increase the risk of missed abortion, and there may be a positive additive interaction between the two factors on missed abortion.

Abstract C030: Relationship between the oral microbiome and tobacco-induced DNA damage in African American and White persons who smoke

Abstract Exposure to tobacco smoke chemicals and toxicants may cause unique changes to an individual’s oral microbiome, impacting immuno-inflammatory responses, the metabolism of tobacco smoke carcinogens, and overall health status. In our previous study, HPB-releasing DNA adducts (DNA damage derived from tobacco-specific nitrosamines) were higher in oral cells of African American (AA, n=75) when compared to White (WH, n=71) persons who smoke, even after adjusting for key confounders such as smoking dose. In this study, we examined the relationship between the levels of HPB-releasing DNA adducts and microbiome composition in these individuals. 16S rRNA gene sequencing, targeting the V4 hypervariable region, was performed on DNA isolated from mouthwash samples. Sequences were clustered into Amplicon Sequence Variants (ASV) using QIIME, and metagenomic content was inferred using PICRUSt. Our results showed a significant difference in alpha-diversity metrics: observed richness and Shannon indexes (Kruskal-Wallis test, H = 8.42, p &amp;lt;0.01 and H = 6.85, p &amp;lt;0.03, respectively) across levels of DNA damage (low, intermediate, and high). Post hoc tests with pairwise comparisons showed that, for both indexes, individuals with high levels of HPB-releasing DNA adducts had significantly lower alpha diversity than those with intermediate levels (p &amp;lt;0.05). However, there were no differences in beta diversity as measured by Bray-Curtis (R2 = 1.15, p = 0.2) and Jaccard (R2 = 1.1, p = 0.1) indices across individuals with different levels of HPB-releasing DNA adducts. Multivariable association tests revealed that high levels of the DNA adducts were positively associated with Enterobacteriaceae, while intermediate levels were associated with Porphyromonas endodontalis, Prevotella spp, Neisseria spp, Actinomyces spp, and Fusobacterium. In addition, predicted functional profiling showed that intermediate levels of HPB-releasing DNA adducts were positively associated with amino acid, carbohydrate, and hem biosynthesis, along with reductive tricarboxylic acid cycle and formaldehyde degradation pathways. Comparisons between AA and WH persons who smoke showed that AA had significantly higher alpha diversity as assessed by observed bacterial richness and The Shannon index (Wilcoxon rank-sum tests, q-value=0.03 and 0.004, respectively). Differences between AA and WH were also found for beta diversity (Bray-Curtis: R2=2.4, p=0.01, Jaccard: R2=1.9, p=0.01, weighted Unifrac R2=0.02 p=0.007). The abundance of taxa associated with intermediate levels of DNA damage (Prevotella spp, Neisseria spp, Actinomyces spp, Fusobacterium) was also higher in AA persons who smoke. Together, our findings suggest that certain compositional and functional characteristics of the oral microbiome may serve as indicators of macromolecular responses to harmful chemical exposures from tobacco. The potential mechanistic contribution of the oral microbiome to such responses and its potential role in the observed racial/ethnic differences in cancer risk due to smoking should be further investigated. Citation Format: Aleksandra Alcheva. Relationship between the oral microbiome and tobacco-induced DNA damage in African American and White persons who smoke [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C030.

Association of polycyclic aromatic hydrocarbons in moss with blood biomarker among nearby residents in Portland, Oregon.

Polycyclic aromatic hydrocarbons (PAHs) are air pollutants that are costly to measure using traditional air-quality monitoring methods. We used an epiphytic bio-indicator (moss genus: Orthotrichum) to cost-effectively evaluate atmospheric deposition of PAHs in Portland, Oregon in May 2013. However, it is unclear if measurements derived from these bioindicators are good proxies for human exposure. To address this question, we simultaneously, measured PAH-DNA adducts in blood samples of non-smokers residing close to the sites of moss measurements. We accounted for individual determinants of PAH uptake that are not related to environmental air quality through questionnaires, e.g., wood fires, consumption of barbecued and fried meats. Spearman rank correlation and linear regression (to control for confounders from the lifestyle factors) evaluated the associations. We did not observe evidence of an association between PAH levels in moss and PAH-DNA adducts in blood of nearby residents (e.g., all correlations p≥0.5), but higher level of adducts were evident in those who used wood fire in their houses in the last 48 hours. It remains to be determined whether bio-indicators in moss can be used for human health risk assessment.

Pathways to Identify Electrophiles In Vivo Using Hemoglobin Adducts: Hydroxypropanoic Acid Valine Adduct and Its Possible Precursors.

Analytical methods and tools for the characterization of the human exposome by untargeted mass spectrometry approaches are advancing rapidly. Adductomics methods have been developed for untargeted screening of short-lived electrophiles, in the form of adducts to proteins or DNA, in vivo. The identification of an adduct and its precursor electrophile in the blood is more complex than that of stable chemicals. The present work aims to illustrate procedures for the identification of an adduct to N-terminal valine in hemoglobin detected with adductomics, and pathways for the tracing of its precursor and possible exposure sources. Identification of the adduct proceeded via preparation and characterization of standards of adduct analytes. Possible precursor(s) and exposure sources were investigated by measurements in blood of adduct formation by precursors in vitro and adduct levels in vivo. The adduct was identified as hydroxypropanoic acid valine (HPA-Val) by verification with a synthesized reference. The HPA-Val was measured together with other adducts (from acrylamide, glycidamide, glycidol, and acrylic acid) in human blood (n = 51, schoolchildren). The HPA-Val levels ranged between 6 and 76 pmol/g hemoglobin. The analysis of reference samples from humans and rodents showed that the HPA-Val adduct was observed in all studied samples. No correlation of the HPA-Val level with the other studied adducts was observed in humans, nor was an increase in tobacco smokers observed. A small increase was observed in rodents exposed to glycidol. The formation of the HPA-Val adduct upon incubation of blood with glycidic acid (an epoxide) was shown. The relatively high adduct levels observed in vivo in relation to the measured reactivity of the epoxide, and the fact that the epoxide is not described as naturally occurring, suggest that glycidic acid is not the only precursor of the HPA-Val adduct identified in vivo. Another endogenous electrophile is suspected to contribute to the in vivo HPA-Val adduct level.

Association of aflatoxin B1 levels with mean CD4 cell count and uptake of ART among HIV infected patients: A prospective study.

Aflatoxin suppresses cellular immunity and accentuates HIV-associated changes in T- cell phenotypes and B- cells. This prospective study was conducted to examine the association of aflatoxin levels with CD4 T-cell count and antiretroviral therapy uptake over time. Sociodemographic and food data were collected from antiretroviral therapy naïve HIV-infected patients. CD4+ counts were collected from participants' medical records. Plasma samples were tested for aflatoxin B1 albumin adducts, hepatitis B surface antigen, and HIV viral load. Participants were separated into high and low aflatoxin groups based on the median aflatoxin B1 albumin adduct level of 10.4 pg/ml for data analysis. Participants with high aflatoxin B1 albumin adduct levels had lower mean CD4 at baseline and at each follow-up period. Adjusted multivariable logistic regression analysis showed that higher baseline aflatoxin B1 adduct levels were associated with statistically significant lower CD4 counts (est = -66.5, p = 0.043). Not starting ART and low/middle socioeconomic status were associated with higher CD4 counts (est = 152.2, p<0.001) and (est = 86.3, p = 0.027), respectively. Consistent correlations of higher aflatoxin B1 adduct levels with lower CD4 over time indicate that there is an independent early and prolonged effect of aflatoxin on CD4 even with the initiation of antiretroviral therapy. The prospective study design, evaluation of baseline and follow-up measures, extensive control for potential confounders, and utilization of objective measures of aflatoxin exposure and CD4 count provide compelling evidence for a strong epidemiologic association that deserves careful attention in HIV care and treatment programs.

DNA damage and health effects in juvenile haddock (Melanogrammus aeglefinus) exposed to PAHs associated with oil-polluted sediment or produced water.

The research objective was to study the presence of DNA damages in haddock exposed to petrogenic or pyrogenic polyaromatic hydrocarbons (PAHs) from different sources: 1) extracts of oil produced water (PW), dominated by 2-ring PAHs; 2) distillation fractions of crude oil (representing oil-based drilling mud), dominated by 3-ring PAHs; 3) heavy pyrogenic PAHs, mixture of 4/5/6-ring PAHs. The biological effect of the different PAH sources was studied by feeding juvenile haddock with low doses of PAHs (0.3–0.7 mg PAH/kg fish/day) for two months, followed by a two-months recovery. In addition to the oral exposure, a group of fish was exposed to 12 single compounds of PAHs (4/5/6-ring) via intraperitoneal injection. The main endpoint was the analysis of hepatic and intestinal DNA adducts. In addition, PAH burden in liver, bile metabolites, gene and protein expression of CYP1A, GST activity, lipid peroxidation, skeletal deformities and histopathology of livers were evaluated. Juvenile haddock responded quickly to both intraperitoneal injection and oral exposure of 4/5/6-ring PAHs. High levels of DNA adducts were detected in livers three days after the dose of the single compound exposure. Fish had also high levels of DNA adducts in liver after being fed with extracts dominated by 2-ring PAHs (a PW exposure scenario) and 3-ring PAHs (simulating an oil exposure scenario). Elevated levels of DNA adducts were observed in the liver of all exposed groups after the 2 months of recovery. High levels of DNA adduct were found also in the intestines of individuals exposed to oil or heavy PAHs, but not in the PW or control groups. This suggests that the intestinal barrier is very important for detoxification of orally exposures of PAHs.

Alterations of mitochondrial bioenergetics, dynamics, and morphology support the theory of oxidative damage involvement in autism spectrum disorder.

Autism spectrum disorder (ASD) has been hypothesized to be a result of the interplay between genetic predisposition and increased vulnerability to early environmental insults. Mitochondrial dysfunctions appear also involved in ASD pathophysiology, but the mechanisms by which such alterations develop are not completely understood. Here, we analyzed ASD primary fibroblasts by measuring mitochondrial bioenergetics, ultrastructural and dynamic parameters to investigate the hypothesis that defects in these pathways could be interconnected phenomena responsible or consequence for the redox imbalance observed in ASD. High levels of 4-hydroxynonenal protein adducts together with increased NADPH (nicotinamide adenine dinucleotide phosphateoxidase) activity and mitochondrial superoxide production coupled with a compromised antioxidant response guided by a defective Nuclear Factor Erythroid 2-Related Factor 2 pathway confirmed an unbalanced redox homeostasis in ASD. Moreover, ASD fibroblasts showed overactive mitochondrial bioenergetics associated with atypical morphology and altered expression of mitochondrial electron transport chain complexes and dynamics-regulating factors. We suggest that many of the changes observed in mitochondria could represent compensatory mechanisms by which ASD cells try to adapt to altered energy demand, possibly resulting from a chronic oxinflammatory status.

Formaldehyde-activated WEHI-150 induces DNA interstrand crosslinks with unique structural features

Mitoxantrone is an anticancer anthracenedione that can be activated by formaldehyde to generate covalent drug-DNA adducts. Despite their covalent nature, these DNA lesions are relatively labile. It was recently established that analogues of mitoxantrone featuring extended side-chains terminating in primary amino groups typically yielded high levels of stable DNA adducts following their activation by formaldehyde. In this study we describe the DNA sequence-specific binding properties of the mitoxantrone analogue WEHI-150 which is the first anthracenedione to form apparent DNA crosslinks mediated by formaldehyde. The utility of this compound lies in the versatility of the covalent binding modes displayed. Unlike other anthracenediones described to date, WEHI-150 can mediate covalent adducts that are independent of interactions with the N-2 of guanine and is capable of adduct formation at novel DNA sequences. Moreover, these covalent adducts incorporate more than one formaldehyde-mediated bond with DNA, thus facilitating the formation of highly lethal DNA crosslinks. The versatility of binding observed is anticipated to allow the next generation of anthracenediones to interact with a broader spectrum of nucleic acid species than previously demonstrated by the parent compounds, thus allowing for more diverse biological activities.

Anti-inflammatory Effect of AZD6244 on Acrolein-Induced Neuroinflammation.

Clinically, high levels of acrolein (a highly reactive α, β-unsaturated aldehyde) and acrolein adducts are detected in the brain of patients with CNS neurodegenerative diseases, including Alzheimer's disease and spinal cord injury. Our previous study supports this notion by showing acrolein as a neurotoxin in a Parkinsonian animal model. In the present study, the effect of AZD6244 (an ATP non-competitive MEK1/2 inhibitor) on acrolein-induced neuroinflammation was investigated using BV-2 cells and primary cultured microglia. Our immunostaining study showed that lipopolysaccharide (LPS, an inflammation inducer as a positive control) increased co-localized immunoreactivities of phosphorylated ERK and ED-1 (a biomarker of activated microglia) in the treated BV-2 cells. Similar elevation in co-localized immunoreactivities of phosphorylated ERK and ED-1 was detected in the acrolein-treated BV-2 cells. Furthermore, Western blot assay showed increases in phosphorylated ERK in BV-2 cells subjected to LPS (1μg/mL) or acrolein (30μM); these increases were blocked by AZD6244 (10μM). At the same time, AZD6244 attenuated LPS-induced TNF-α (a pro-inflammatory cytokine) and cyclooxygenase-II (COX II, a pro-inflammatory enzyme). Consistently, AZD6244 reduced acrolein-induced elevations in COX-II mRNA and COX-II protein expression. In addition, AZD6244 inhibited acrolein-induced increases in activated caspase 1 (a biomarker of inflammasome activation) and heme oxygenase-1 (a redox-regulated chaperone protein) in BV-2 cells. Using a transwell migration assay, AZD6244 attenuated acrolein (5μM)-induced migration of BV-2 cells and primary cultured microglia. In conclusion, our study shows that acrolein is capable of inducing neuroinflammation which involved ERK activation in microglia. Furthermore, AZD6244 is capable of inhibiting acrolein-induced neuroinflammation. Our study suggests that ERK inhibition may be a neuroprotective target against acrolein-induced neuroinflammation in the CNS neurodegenerative diseases.

1-Methoxy-3-indolylmethyl DNA adducts in six tissues, and blood protein adducts, in mice under pak choi diet: time course and persistence.

We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N2-(1-MIM)-dG, N6-(1-MIM)-dA] in six tissues, as well as protein adducts [τN-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC-MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.

The Link between Oxidative Stress and Cancer: Prevention through Yoga

Sperm DNA damage induced by oxidative stress may lead to accumulation of mutations in sperm genome due to the presence of mutagenic oxidative adducts in DNA. This may contribute to the development of male infertility, recurrent pregnancy loss, congenital malformations, neurodevelopmental disorders, and childhood cancers. This article highlights the impact of lifestyle habits of father preconceptionally, its lifelong impact on offspring’s health and impact of yoga-based lifestyle intervention on sperm genome and epigenome. Sperm is transcriptionally and translationally inert with a very basic repair mechanism and is thus unable to repair DNA damage and this may persist postfertilization and lead to accumulation of damaged DNA in each cell of zygote. This DNA damage with high levels of mutagenic oxidative base adducts may lead to accumulation of mutations in both sperm DNA (germline) and zygote (somatic). Majority of mutations arise during cell replication and as sperm has a limited capacity for DNA damage detection and repair, it is highly susceptible to accumulation of mutations. Paternal unhealthy lifestyle habits like smoking, alcohol, or tobacco consumption are positively associated with the risk of childhood cancers like nonfamilial sporadic heritable retinoblastoma. Lifestyle interventions like yoga improve sperm DNA integrity by reducing levels of oxidative DNA damage, optimizing oxidative stress, and by increasing the expression of genes responsible for DNA repair, cell-cycle control, and anti-inflammatory effects. Thus, social habits affect sperm DNA health and as such, it is recommended that simple lifestyle modifications (diet rich in fruits and vegetables, yoga, meditation) improve DNA health and may thereby decrease the incidence of childhood morbidity and cancer. Hence, biological parenting commences well before birth and even prior to conception, which highlights the need for fathers to adopt a healthy lifestyle.

Intestinal polycyclic aromatic hydrocarbon-DNA adducts in a population of beluga whales with high levels of gastrointestinal cancers.

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) were disposed directly into the Saguenay River of the St. Lawrence Estuary (SLE) by local aluminum smelters (Quebec, Canada) for 50 years (1926-1976). PAHs in the river sediments are likely etiologically related to gastrointestinal epithelial cancers observed in 7% of 156 mature (>19-year old) adult beluga found dead along the shorelines. Because DNA adduct formation provides a critical link between exposure and cancer induction, and because PAH-DNA adducts are chemically stable, we hypothesized that SLE beluga intestine would contain PAH-DNA adducts. Using an antiserum specific for DNA modified with several carcinogenic PAHs, we stained sections of paraffin-embedded intestine from 51 SLE beluga (0-63 years), 4 Cook Inlet (CI) Alaska beluga (0-26 years), and 20 beluga (0-46 years) living in Arctic areas (Eastern Beaufort Sea, Eastern Chukchi Sea, Point Lay Alaska) and aquaria, all with low PAH contamination. Stained sections showed nuclear light-to-dark pink color indicating the presence of PAH-DNA adducts concentrated in intestinal crypt epithelial lining cells. Scoring of whole tissue sections revealed higher values for the 51 SLE beluga, compared with the 20 Arctic and aquarium beluga (P = 0.003). The H-scoring system, applied to coded individual photomicrographs, confirmed that SLE beluga and CI beluga had levels of intestinal PAH-DNA adducts significantly higher than Arctic and aquarium beluga (P = 0.003 and 0.02, respectively). Furthermore, high levels of intestinal PAH-DNA adducts in four SLE beluga with gastrointestinal cancers, considered as a group, support a link of causality between PAH exposure and intestinal cancer in SLE beluga. Environ. Mol. Mutagen. 60:29-41, 2019. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Outbreak of an acute aflatoxicosis in Tanzania during 2016

In June 2016, an outbreak of an unknown disease was reported to affect clusters of families in two regions of the central part of Tanzania. A rapid epidemiological survey was conducted in the affected villages, with a detailed house-to-house survey in selected households. A total of 68 cases occurred between 14 May and 14 November 2016, of which 20 died, making a case fatality rate of 30%. Over 50% of the cases were below the age of 15 years. The cases presented with jaundice (n=60), abdominal pain (n=59), vomiting (n=56), diarrhoea (n=34) and ascites (n=32). The responsible food item appeared to be home grown maize. The rate ratio indicated that the occurrence of illnesses was associated with ingestion of food contaminated with high levels of aflatoxins (contamination range: 10-51,100 μg/kg and 2.4-285 μg/kg for case and control households, respectively). Serum aflatoxin biomarker indicated that cases were more likely to have higher than 1000 pg/mg aflatoxin-albumin adduct level in their sera compared to controls (Odds Ratio = 13.5; 95% confidence intervals = 1.5-165.3; range of aflatoxin-albumin adduct level = 36- 32,800 pg/mg for cases and 10-4020 pg/mg for controls). Beside aflatoxins, maize samples were also contaminated with high levels of fumonisins (range of contamination; 945-12,630 μg/kg) with 8 of 10 samples analysed from case households co-contaminated with both toxins at levels above the maximum limit of 5 or 10 μg/kg set for AFB1 or total aflatoxins and 2,000 μg/kg for fumonisins. Clinical presentation and high levels of aflatoxin in food samples coupled with high levels of serum aflatoxin-albumin adducts among the cases support the causal role of aflatoxins.

Biochemical Mechanism of Rhododendrol-Induced Leukoderma.

RS-4-(4-hydroxyphenyl)-2-butanol (rhododendrol (RD))—a skin-whitening ingredient—was reported to induce leukoderma in some consumers. We have examined the biochemical basis of the RD-induced leukoderma by elucidating the metabolic fate of RD in the course of tyrosinase-catalyzed oxidation. We found that the oxidation of racemic RD by mushroom tyrosinase rapidly produces RD-quinone, which gives rise to secondary quinone products. Subsequently, we confirmed that human tyrosinase is able to oxidize both enantiomers of RD. We then showed that B16 cells exposed to RD produce high levels of RD-pheomelanin and protein-SH adducts of RD-quinone. Our recent studies showed that RD-eumelanin—an oxidation product of RD—exhibits a potent pro-oxidant activity that is enhanced by ultraviolet-A radiation. In this review, we summarize our biochemical findings on the tyrosinase-dependent metabolism of RD and related studies by other research groups. The results suggest two major mechanisms of cytotoxicity to melanocytes. One is the cytotoxicity of RD-quinone through binding with sulfhydryl proteins that leads to the inactivation of sulfhydryl enzymes and protein denaturation that leads to endoplasmic reticulum stress. The other mechanism is the pro-oxidant activity of RD-derived melanins that leads to oxidative stress resulting from the depletion of antioxidants and the generation of reactive oxygen radicals.

Effect of Benzo[a]Pyrene on Spindle Misorientation and Fidelity of Chromosome Segregation in Lung Epithelial BEAS-2B Cells.

Benzo[a]pyrene (B[a]P) is an environmental carcinogen found in tobacco smoke. It leads to high levels of DNA adducts in the lungs of cigarette smokers contributing to genomic instability. Alterations in the mitotic spindle apparatus play a major role in the generation of genomic instability through promoting chromosome mis-segregation and aneuploidy. To date, the effect of B[a]P exposure on altering the mitotic apparatus in normal lung epithelial cells remains unknown. In our study, BEAS-2B human bronchial epithelial cells were exposed to B[a]P and spindle dynamics were evaluated. Confocal imaging showed that B[a]P exposure significantly alters spindles misorientation, leading to chromosome mis-segregations in the form of chromosome lags and bridges. In addition, centrosome duplication and premature centriole disengagement were induced leading to misaligned and multipolar spindle formation. Comparative genomic analysis of mitotic spindle associated genes, revealed downregulation of AurA-Plk1-AurB signaling cascade by B[a]P. In addition, we analyzed the status of p53 and its downstream p21 in B[a]P-treated cells and showed a suppression of p53-p21 axis. When the extent of DNA damage associated with induced mitotic abnormalities was investigated using γ-H2AX, a significant increase and persistence in DNA damage was observed. Overall, our findings show that B[a]P potently induces mitotic abnormalities, DNA damage, and genetic instability.

Highlights of This Issue

The pharmacological inhibition of lysine specific demethylase 1 (LSD1, also known as KDM1A) has recently drawn attention as a novel therapeutic option against acute myeloid leukemia (AML). Here, Ishikawa, Gamo, and colleagues demonstrated that a novel irreversible LSD1 inhibitor, T-3775440, exhibited anti-leukemic efficacy in GFI1B-expressing acute erythroleukemia (AEL) and acute megakaryoblastic leukemia (AMKL) cells. T-3775440 exerts anti-AML effects through a mechanism involving disruption of the LSD1/GFI1B complex and consequent AML cell transdifferentiation. Given that several LSD1 inhibitors have entered clinical trials for treating patients with AML, these results provide new perspectives in the therapeutic strategy of AEL and AMKL.The mechanism of acquired resistance against afatinib is still unclear. Here, Kobayashi and colleagues established a panel of resistant clones from Ba/F3 cells transduced with mutated EGFR genes (Del18, G719A, Del19 or L858R). Among 84 resistant clones, secondary C797S mutation was found in subsets of Del18, G719A or L858R but not in Del19 clones. A novel L792F was present in Del18 clones while the majority had a common T790M. Clones with C797S and L792F exhibit sensitivity to erlotinib and dacomitinib, respectively. In conclusion, afatinib resistance could be driven by L792F or C797S mutations and therefore testing novel combinations of EGFR-TKIs might benefit patients with these minor mutations.Common cytotoxic chemotherapy regimens currently lack diagnostic tests that predict response. Zimmermann and colleagues demonstrated the feasibility of using microdose-induced drug-DNA adducts as predictive biomarkers of response to gemcitabine and cisplatin or carboplatin in a pilot clinical study and several mouse models of bladder cancer. Patients with the highest carboplatin-DNA adduct levels were responders, but not all responders had high adduct levels. In mice bearing patient-derived tumor xenografts, at least one of the drugs in the regimen had to induce high drug-DNA adduct levels or a synergistic increase in overall adducts to prompt a corresponding therapeutic response.The potential of epigenetic drugs to synergize with other drugs against colon cancer cells remains to be uncovered. To identify new drug interactions, high-throughput screening was performed on cancer cells treated with DNA methylation or histone deacetylase inhibitors in combination with FDA-approved drug libraries. The screening model measured reporter gene expression level, as a surrogate for tumor suppressor gene reactivation. This study revealed new combinations of epigenetic drugs and FDA-approved drugs producing epigenetic synergy, which can be rapidly implemented into clinical trials against colon cancer.

Microdose-Induced Drug-DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice.

We report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [14C]carboplatin (1% of the therapeutic dose). Carboplatin-DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice were dosed with [14C]carboplatin or [14C]gemcitabine and the resulting drug-DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug-DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug-DNA adducts as predictive biomarkers. Mol Cancer Ther; 16(2); 376-87. ©2016 AACR.

Etiology and prevention of prevalent types of cancer.

Endogenous estrogens become carcinogens when excessive catechol estrogen quinone metabolites are formed. Specifically, the catechol estrogen-3,4-quinones can react with DNA to produce a large amount of specific depurinating estrogen-DNA adducts, formed at the N-3 of Ade and N-7 of Gua. Loss of these adducts leaves apurinic sites in the DNA, which can generate subsequent cancer-initiating mutations. Unbalanced estrogen metabolism yields excessive catechol estrogen-3,4-quinones, increasing formation of the depurinating estrogen-DNA adducts and the risk of initiating cancer. Evidence for this mechanism of cancer initiation comes from studies in vitro, in cell culture, in animal models and in human subjects. High levels of estrogen-DNA adducts have been observed in women with breast, ovarian or thyroid cancer, and in men with prostate cancer or non-Hodgkin lymphoma. Observation of high levels of depurinating estrogen-DNA adducts in high risk women before the presence of breast cancer indicates that adduct formation is a critical factor in breast cancer initiation. Two dietary supplements, N-acetylcysteine and resveratrol, complement each other in reducing formation of catechol estrogen-3,4-quinones and inhibiting formation of estrogen-DNA adducts in cultured human and mouse breast epithelial cells. They also inhibit malignant transformation of these epithelial cells. In addition, formation of adducts was reduced in women who followed a Healthy Breast Protocol that includes N-acetylcysteine and resveratrol. Blocking initiation of cancer prevents promotion, progression and development of the disease. These results suggest that reducing formation of depurinating estrogen-DNA adducts can reduce the risk of developing a variety of types of human cancer.

Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549).

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.