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Background: Cancer hazard identification is critical to informing decisions on preventive actions. However, the influence of cancer hazard assessments on the creation of health-protective regulations is poorly understood. Although prior studies have measured the health and economic benefits of regulatory actions in general, we are not aware of efforts to explicitly study the influence of cancer hazard identification on policy decisions in the United States., Objectives: In this commentary, we present an approach to examine whether formal identification of a substance as a human carcinogen may prompt a regulatory action to reduce exposure to carcinogens and enhance public health. Further, we discuss the broader implications of cancer hazard identification on policy decision-making, including identifying gaps and providing recommendations., Methods: Using the Report on Carcinogens (RoC) as a test case, we systematically searched U.S. federal and state databases for notices of regulations mentioning the RoC from 1995 to 2023. For each regulation, we extracted information on the carcinogen(s) regulated, the regulatory agency, the regulatory purpose, the economic sector exposure sources, and the analyzed public health benefits and costs. We created a publicly available, web-based interactive tool to visualize the data., Discussion: U.S. regulatory agencies have been using cancer hazard evaluations, such as the RoC, for decades to inform public health policy actions to prevent or mitigate cancer risks. Specifically, nonregulatory cancer hazard assessments have been used to prioritize chemical evaluations, support regulatory-based assessments, and trigger regulatory action. Our approach showed that assessing the influence of cancer hazard identification on science-based public health policies is feasible, informative, and needed, and our study is a first step in this direction. We recommend expanding this approach to other cancer and noncancer hazard assessments to ultimately inform our understanding of the influence of hazard classifications on policymaking. https://doi.org/10.1289/EHP12681.

Background and Aim: Epidemiological studies conducted mostly in low- and middle-income countries have found a positive association between household combustion of wood and lung cancer. However, most studies have been retrospective, and few have been conducted in the United States where indoor wood-burning usage patterns differ. We examined the association of exposure to indoor wood smoke from fireplaces and stoves with incident lung cancer in a U.S.-wide cohort of women., Methods: We included 50,226 women without prior lung cancer participating in the U.S.-based prospective Sister Study. At enrollment (2003-2009), women reported frequency of use of wood-burning stoves and/or fireplaces in their longest-lived adult residence. Cox regression was used to estimate adjusted hazard ratios (HR adj ) and 95 % confidence intervals (CI) for the association between indoor wood-burning fireplace/stove use and incident lung cancer. Lung cancer was self-reported and confirmed with medical records., Results: During an average 11.3 years of follow-up, 347 medically confirmed lung cancer cases accrued. Overall, 62.3 % of the study population reported the presence of an indoor wood-burning fireplace/stove at their longest-lived adult residence and 20.6 % reported annual usage of ≥30 days/year. Compared to those without a wood-burning fireplace/stove, women who used their wood-burning fireplace/stove ≥30 days/year had an elevated rate of lung cancer (HR adj  = 1.68; 95 % CI = 1.27, 2.20). In never smokers, positive associations were seen for use 1-29 days/year (HR adj  = 1.64; 95 % CI = 0.87, 3.10) and ≥30 days/year (HR adj  = 1.99; 95 % CI = 1.02, 3.89). Associations were also elevated across all income groups, in Northeastern, Western or Midwestern U.S. regions, and among those who lived in urban or rural/small town settings., Conclusions: Our prospective analysis of a cohort of U.S. women found that increasing frequency of wood-burning indoor fireplace/stove usage was associated with incident lung cancer, even among never smokers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The National Toxicology Program strives to raise awareness of cancer hazards in our environment. Identifying cancer hazards is key to primary prevention, informing public health decision making, and decreasing the global cancer burden. In December 2021, the US congressionally mandated 15th Report on Carcinogens was released, adding 8 new substances to the cumulative report. Chronic infection with Helicobacter pylori is listed as "known to be a human carcinogen." Antimony trioxide and 6 haloacetic acids found as water disinfection by-products-dichloroacetic acid, dibromoacetic acid, bromochloroacetic acid, tribromoacetic acid, bromodichloroacetic acid, chlorodibromoacetic acid-are listed as "reasonably anticipated to be a human carcinogen." A new dashboard provides interactive visualization and interrogation of the 256 listed substances, their uses, and associated cancers. Also, the National Toxicology Program recently published a Cancer Hazard Assessment Report on exposure scenarios associated with circadian disruption, concluding that persistent night shift work can cause breast cancer and certain lighting conditions may cause cancer. As highlighted in these reports and evaluations, we are evolving our approaches to meet contemporary challenges. These approaches include focusing on real-world exposures and advancing our methods to address challenges in cancer hazard assessments (eg, developing more structured approaches to evaluate mechanistic data and incorporating read-across approaches to assess chemicals lacking adequate human or animal cancer data). To promote public health, we provide information on environmental health disparities and disease prevention. Building on these efforts, we aim to continue our contributions to the war on cancer, declared 50 years ago., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This commentary is written in response to a recent commentary in Carcinogenesis that provides several viewpoints on the International Agency for Research on Cancer's (IARC) Monographs program on cancer hazard identification. This commentary offers an alternative viewpoint of the role of cancer hazard identification derived from cancer epidemiology studies in risk characterization, as well as clarification on the previous commentary's interpretation of the purpose of the Monographs and other programs of cancer hazard identification and how IARC communicates the findings of the Monographs., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Harmonized language is critical for helping researchers to find data, collecting scientific data to facilitate comparison, and performing pooled and meta-analyses. Using standard terms to link data to knowledge systems facilitates knowledge-driven analysis, allows for the use of biomedical knowledge bases for scientific interpretation and hypothesis generation, and increasingly supports artificial intelligence (AI) and machine learning. Due to the breadth of environmental health sciences (EHS) research and the continuous evolution in scientific methods, the gaps in standard terminologies, vocabularies, ontologies, and related tools hamper the capabilities to address large-scale, complex EHS research questions that require the integration of disparate data and knowledge sources. The results of prior workshops to advance a harmonized environmental health language demonstrate that future efforts should be sustained and grounded in scientific need. We describe a community initiative whose mission was to advance integrative environmental health sciences research via the development and adoption of a harmonized language. The products, outcomes, and recommendations developed and endorsed by this community are expected to enhance data collection and management efforts for NIEHS and the EHS community, making data more findable and interoperable. This initiative will provide a community of practice space to exchange information and expertise, be a coordination hub for identifying and prioritizing activities, and a collaboration platform for the development and adoption of semantic solutions. We encourage anyone interested in advancing this mission to engage in this community.

Background: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic compounds associated with chronic disease in epidemiologic studies, though the contribution of PAH exposure on fatal outcomes in the U.S. is largely unknown., Objectives: We investigated urinary hydroxylated PAH metabolites (OH-PAHs) with all-cause and cause-specific mortality in a representative sample of the U.S. population., Methods: Study participants were ≥20 years old from the National Health and Nutrition Examination Survey 2001-2014. Concentrations (nmol/L) of eight OH-PAHs from four parent PAHs (naphthalene, fluorene, phenanthrene, pyrene) were measured in spot urine samples at examination. We identified all-cause, cancer-specific, and cardiovascular-specific deaths through 2015 using the National Death Index. We used Cox proportional hazards regression to estimate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between ΣOH-PAHs and mortality endpoints. We assessed potential heterogeneity by age, gender, smoking status, poverty, and race/ethnicity. Additionally, we examined the overall mixture effect using quantile g-computation., Results: In 9,739 eligible participants, there were 934 all-cause deaths, 159 cancer-specific deaths, and 108 cardiovascular-specific deaths (median 6.75 years follow-up). A log10 increase in ΣOH-PAHs was associated with higher all-cause mortality (HRadj = 1.39 [95%CI: 1.21, 1.61]), and possibly cancer-specific mortality (HRadj = 1.15 [95%CI: 0.79, 1.69]), and cardiovascular-specific mortality (HRadj = 1.49 [95%CI: 0.94, 2.33]). We observed substantial effect modification by age, smoking status, gender, and race/ethnicity across mortality endpoints. Risk of cardiovascular mortality was higher for non-Hispanic blacks and those in poverty, indicating potential disparities. Quantile g-computation joint associations for a simultaneous quartile increase in OH-PAHs were HRadj = 1.15 [95%CI: 1.02, 1.31], HRadj = 1.41 [95%CI: 1.05, 1.90], and HRadj = 0.98 [95%CI: 0.66, 1.47] for all-cause, cancer-specific, and cardiovascular-specific mortalities, respectively., Discussion: Our results support a role for total PAH exposure in all-cause and cause-specific mortality in the U.S. population., Competing Interests: The authors have declared that no competing interests exist.

Background: Over 800 pesticides are registered for use in the United States. Human studies indicate concern that some pesticides currently in use in large quantities may also pose a carcinogenic hazard. Our objective is to identify candidates for future hazard evaluations among pesticides used in high volumes in the United States and also classified as potential carcinogens by U.S. Environmental Protection Agency (USEPA). We also identify data gaps where further research is needed., Methods: We used a systematic, two-tiered review approach to prioritize pesticides. First, we identified currently registered pesticides classified by USEPA as "possible", "suggestive", or "likely" human carcinogens. Among these, we selected pesticides USEPA has listed as commonly used by volume in at least one sector (agriculture, home and garden, or industry, commercial, and/or government), and those without a published hazard evaluation in the past 5 years. Second, we searched primary literature databases for peer-reviewed human cancer studies reporting pesticide-specific data published since the last USEPA carcinogenicity evaluation for each pesticide, and created evidence maps of the number of studies meeting our criteria for each identified pesticide. No evaluation of study results or risk-of-bias assessments were conducted., Results: We identified 18 pesticides meeting our selection criteria, 16 pesticides had information from human cancer studies published after their initial carcinogenicity review. Of these, eight pesticides had at least three studies for one or more cancer sites: carbaryl, dichloropropene, dimethoate, mancozeb, metolachlor, pendimethalin, permethrin, and trifluralin. A major limitation in the literature revealed a shortage of studies reporting risk estimates for individual pesticides, rather pesticides were grouped by chemical class., Conclusions: Our scoping report provides a map of the existing literature on real-world exposures and human cancer that has accumulated on pesticides classified as potential carcinogens by USEPA and used in high volumes. We also illustrate that several pesticides which are "data-rich" may warrant updated authoritative hazard evaluations. Our two-tiered approach and utilization of evidence mapping can be used to inform future decision-making to update cancer hazard evaluations.

Systematic reviews are powerful tools for drawing causal inference for evidence-based decision-making. Published systematic reviews and meta-analyses of environmental and occupational epidemiology studies have increased dramatically in recent years; however, the quality and utility of published reviews are variable. Most methodologies were adapted from clinical epidemiology and have not been adequately modified to evaluate and integrate evidence from observational epidemiology studies assessing environmental and occupational hazards, especially in evaluating the quality of exposure assessments. Although many reviews conduct a systematic and transparent assessment for the potential for bias, they are often deficient in subsequently integrating across a body of evidence. A cohesive review considers the impact of the direction and magnitude of potential biases on the results, systematically evaluates important scientific issues such as study sensitivity and effect modifiers, identifies how different studies complement each other, and assesses other potential sources of heterogeneity. Given these challenges of conducting informative systematic reviews of observational studies, we provide a series of specific recommendations based on practical examples for cohesive evidence integration to reach an overall conclusion on a body of evidence to better support policy making in public health.

Background: Increasingly, risk of bias tools are used to evaluate epidemiologic studies as part of evidence synthesis (evidence integration), often involving meta-analyses. Some of these tools consider hypothetical randomized controlled trials (RCTs) as gold standards., Methods: We review the strengths and limitations of risk of bias assessments, in particular, for reviews of observational studies of environmental exposures, and we also comment more generally on methods of evidence synthesis., Results: Although RCTs may provide a useful starting point to think about bias, they do not provide a gold standard for environmental studies. Observational studies should not be considered inherently biased vs. a hypothetical RCT. Rather than a checklist approach when evaluating individual studies using risk of bias tools, we call for identifying and quantifying possible biases, their direction, and their impacts on parameter estimates. As is recognized in many guidelines, evidence synthesis requires a broader approach than simply evaluating risk of bias in individual studies followed by synthesis of studies judged unbiased, or with studies given more weight if judged less biased. It should include the use of classical considerations for judging causality in human studies, as well as triangulation and integration of animal and mechanistic data., Conclusions: Bias assessments are important in evidence synthesis, but we argue they can and should be improved to address the concerns we raise here. Simplistic, mechanical approaches to risk of bias assessments, which may particularly occur when these tools are used by nonexperts, can result in erroneous conclusions and sometimes may be used to dismiss important evidence. Evidence synthesis requires a broad approach that goes beyond assessing bias in individual human studies and then including a narrow range of human studies judged to be unbiased in evidence synthesis. https://doi.org/10.1289/EHP6980.

The Monographs produced by the International Agency for Research on Cancer (IARC) apply rigorous procedures for the scientific review and evaluation of carcinogenic hazards by independent experts. The Preamble to the IARC Monographs, which outlines these procedures, was updated in 2019, following recommendations of a 2018 expert advisory group. This article presents the key features of the updated Preamble, a major milestone that will enable IARC to take advantage of recent scientific and procedural advances made during the 12 years since the last Preamble amendments. The updated Preamble formalizes important developments already being pioneered in the Monographs program. These developments were taken forward in a clarified and strengthened process for identifying, reviewing, evaluating, and integrating evidence to identify causes of human cancer. The advancements adopted include the strengthening of systematic review methodologies; greater emphasis on mechanistic evidence, based on key characteristics of carcinogens; greater consideration of quality and informativeness in the critical evaluation of epidemiological studies, including their exposure assessment methods; improved harmonization of evaluation criteria for the different evidence streams; and a single-step process of integrating evidence on cancer in humans, cancer in experimental animals, and mechanisms for reaching overall evaluations. In all, the updated Preamble underpins a stronger and more transparent method for the identification of carcinogenic hazards, the essential first step in cancer prevention., (© The Author(s) 2019. Published by Oxford University Press.)

Background: Red and processed meats have been implicated as risk factors in the development of colorectal cancer in U.S. women, but associations with cooking practices are less well established., Methods: Data are from the Sister Study, a cohort of women ages 35 to 74 years from the United States and Puerto Rico who have a sister diagnosed with breast cancer. Red and processed meat consumption, meat cooking practices, and intake of common meat products were collected at baseline using self-administered questionnaires ( N = 48,704). Multivariable HRs (HR adj ) and 95% confidence intervals (95% CI) were estimated., Results: During a median 8.7 years' follow-up (range <1-12.7 years), 216 colorectal cancer cases were diagnosed. In categorical analyses, an increased risk of colorectal cancer was seen in the highest quartile of processed meat consumption compared with the lowest [HR adj = 1.52 (95% CI, 1.01-2.30); P trend = 0.02], and for specific meat products, including breakfast sausages [HR adj = 1.85 (95% CI, 1.30-2.64)] and bacon [HR adj = 1.46 (95% CI, 1.01-2.11)]. The HR adj for the highest quartile of red meat consumption was 1.04 (95% CI, 0.68-1.60), and little evidence of association was observed for cooking practices or doneness of red meat. We observed positive associations with specific red meat products when cooking methods were considered, for example, grilled/barbequed steaks [HR adj = 2.23 (95% CI, 1.20-4.14)] and hamburgers [HR adj = 1.98 (95% CI, 1.00-3.91)]., Conclusions: Higher reported daily intake of processed meats and consumption of barbecued/grilled red meat products were associated with increased risk of colorectal cancer in women., Impact: Variability in colorectal risk risk by meat type and cooking method should be considered when evaluating meat consumption., (©2019 American Association for Cancer Research.)

Background: Due to the large number of chemicals not yet tested for carcinogenicity but to which people are exposed, the limited number of human and animal cancer studies conducted each year, and the frequent need for a timely response, mechanistic data are playing an increasingly important role in carcinogen hazard identification., Objectives: To provide a targeted approach to identify relevant mechanistic data in our cancer evaluation of haloacetic acids (HAAs), we used several approaches including systematic review, the 10 key characteristics of carcinogens (KCs), and read-across methods. Our objective in this commentary is to discuss the strengths, limitations, and challenges of these approaches in a cancer hazard assessment., Methods: A cancer hazard assessment for 13 HAAs found as water disinfection by-products was conducted. Literature searches for mechanistic studies focused on the KCs and individual HAAs. Studies were screened for relevance and categorized by KCs and other relevant data, including chemical properties, toxicokinetics, and biological effects other than KCs. Mechanistic data were organized using the KCs, and strength of evidence was evaluated; this information informed potential modes of action (MOAs) and read-across-like approaches. Three read-across options were considered: evaluating HAAs as a class, as subclass(es), or as individual HAAs (analog approach)., Discussion: Because of data limitations and uncertainties, listing as a class or subclass(es) was ruled out, and an analog approach was used. Two brominated HAAs were identified as target (untested) chemicals based on their metabolism and similarity to source (tested) chemicals. In addition, four HAAs with animal cancer data had sufficient evidence for potential listing in the Report on Carcinogens (RoC). This is the first time that the KCs and other relevant data, in combination with read-across principles, were used to support a recommendation to list chemicals in the RoC that did not have animal cancer data. https://doi.org/10.1289/EHP5672.

Background: An estimated 110 million workers are exposed to welding fumes worldwide. Welding fumes are classified by the International Agency for Research on Cancer as carcinogenic to humans (group 1), based on sufficient evidence of lung cancer from epidemiological studies., Objective: To conduct a meta-analysis of case-control and cohort studies on welding or exposure to welding fumes and risk of lung cancer, accounting for confounding by exposure to asbestos and tobacco smoking., Methods: The literature was searched comprehensively in PubMed, reference lists of relevant publications and additional databases. Overlapping populations were removed. Meta-relative risks (mRRs) were calculated using random effects models. Publication bias was assessed using funnel plot, Eggers's test and Begg's test., Results: Forty-five studies met the inclusion criteria (20 case-control, 25 cohort/nested case-control), which reduced to 37 when overlapping study populations were removed. For 'ever' compared with 'never' being a welder or exposed to welding fumes, mRRs and 95% CIs were 1.29 (1.20 to 1.39; I 2 =26.4%; 22 studies) for cohort studies, 1.87 (1.53 to 2.29; I 2 =44.1%; 15 studies) for case-control studies and 1.17 (1.04 to 1.38; I 2 =41.2%) for 8 case-control studies that adjusted for smoking and asbestos exposure. The mRRs were 1.32 (95% CI 1.20 to 1.45; I 2 =6.3%; 15 studies) among 'shipyard welders', 1.44 (95% CI 1.07 to 1.95; I 2 =35.8%; 3 studies) for 'mild steel welders' and 1.38 (95% CI 0.89 to 2.13; I 2 =68.1%; 5 studies) among 'stainless steel welders'. Increased risks persisted regardless of time period, geographic location, study design, occupational setting, exposure assessment method and histological subtype., Conclusions: These results support the conclusion that exposure to welding fumes increases the risk of lung cancer, regardless of the type of steel welded, the welding method (arc vs gas welding) and independent of exposure to asbestos or tobacco smoking., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective and systematic methods to search, review, and synthesize published studies are a fundamental aspect of carcinogen hazard classification. Systematic review is a historical strength of the International Agency for Research on Cancer (IARC) Monographs Program and the United States National Toxicology Program (NTP) Office of the Report on Carcinogens (RoC). Both organizations are tasked with evaluating peer-reviewed, published evidence to determine whether specific substances, exposure scenarios, or mixtures pose a cancer hazard to humans. This evidence synthesis is based on objective, transparent, published methods that call for extracting and interpreting data in a systematic manner from multiple domains, including a ) human exposure, b ) epidemiological evidence, c ) evidence from experimental animals, and d ) mechanistic evidence. The process involves multiple collaborators and requires an extensive literature search, review, and synthesis of the evidence. Several online tools have been implemented to facilitate these collaborative systematic review processes. Specifically, Health Assessment Workplace Collaborative (HAWC) and Table Builder are custom solutions designed to record and share the results of the systematic literature search, data extraction, and analyses. In addition, a content management system for web-based project management and document submission has been adopted to enable access to submitted drafts simultaneously by multiple co-authors and to facilitate their peer review and revision. These advancements in cancer hazard classification have applicability in multiple systematic review efforts. https://doi.org/10.1289/EHP4224.

The invention of electric light has facilitated a society in which people work, sleep, eat, and play at all hours of the 24-hour day. Although electric light clearly has benefited humankind, exposures to electric light, especially light at night (LAN), may disrupt sleep and biological processes controlled by endogenous circadian clocks, potentially resulting in adverse health outcomes. Many of the studies evaluating adverse health effects have been conducted among night- and rotating-shift workers, because this scenario gives rise to significant exposure to LAN. Because of the complexity of this topic, the National Toxicology Program convened an expert panel at a public workshop entitled "Shift Work at Night, Artificial Light at Night, and Circadian Disruption" to obtain input on conducting literature-based health hazard assessments and to identify data gaps and research needs. The Panel suggested describing light both as a direct effector of endogenous circadian clocks and rhythms and as an enabler of additional activities or behaviors that may lead to circadian disruption, such as night-shift work and atypical and inconsistent sleep-wake patterns that can lead to social jet lag. Future studies should more comprehensively characterize and measure the relevant light-related exposures and link these exposures to both time-independent biomarkers of circadian disruption and biomarkers of adverse health outcomes. This information should lead to improvements in human epidemiological and animal or in vitro models, more rigorous health hazard assessments, and intervention strategies to minimize the occurrence of adverse health outcomes due to these exposures., (Published by Elsevier B.V.)

A viral etiology of cancer was first demonstrated in 1911 by Peyton Rous who injected cell-free filtrate from a chicken sarcoma into healthy chickens and found it induced a tumour. Since the discovery over 50 years ago of the Epstein-Barr virus as the cause of Burkitt lymphoma, seven other human viruses or groups of viruses-hepatitis B virus, hepatitis C virus, human immunodeficiency virus type 1, some human papillomaviruses, human T-cell lymphotropic virus type 1, Kaposi sarcoma-associated herpesvirus and Merkel cell polyomavirus-have been linked to human cancer. Collectively, these eight viruses cause over 20 different types of cancer and contribute to 10-12% of all cancer, with a greater burden in low- and middle-income countries. For many viruses, immunosuppression greatly increases the risks of persistent infection, development of chronic sequelae and cancer. Although several viruses share similar routes of transmission (especially sexual activity, injection drug use and mother-to-child transmission), the predominant route of transmission varies across viruses, and for the same virus can vary by geographical location. In general, vulnerable populations at the greatest risk for viral infections and their associated diseases include people, especially children, living in low- to middle-income countries, men who have sex with men, people who use injection drugs and female sex workers.This article is part of the themed issue 'Human oncogenic viruses'., (© 2017 The Author(s).)

A critical step in systematic reviews of potential health hazards is the structured evaluation of the strengths and weaknesses of the included studies; risk of bias is a term often used to represent this process, specifically with respect to the evaluation of systematic errors that can lead to inaccurate (biased) results (i.e. focusing on internal validity). Systematic review methods developed in the clinical medicine arena have been adapted for use in evaluating environmental health hazards; this expansion raises questions about the scope of risk of bias tools and the extent to which they capture the elements that can affect the interpretation of results from environmental and occupational epidemiology studies and in vivo animal toxicology studies, (the studies typically available for assessment of risk of chemicals). One such element, described here as "sensitivity", is a measure of the ability of a study to detect a true effect or hazard. This concept is similar to the concept of the sensitivity of an assay; an insensitive study may fail to show a difference that truly exists, leading to a false conclusion of no effect. Factors relating to study sensitivity should be evaluated in a systematic manner with the same rigor as the evaluation of other elements within a risk of bias framework. We discuss the importance of this component for the interpretation of individual studies, examine approaches proposed or in use to address it, and describe how it relates to other evaluation components. The evaluation domains contained within a risk of bias tool can include, or can be modified to include, some features relating to study sensitivity; the explicit inclusion of these sensitivity criteria with the same rigor and at the same stage of study evaluation as other bias-related criteria can improve the evaluation process. In some cases, these and other features may be better addressed through a separate sensitivity domain. The combined evaluation of risk of bias and sensitivity can be used to identify the most informative studies, to evaluate the confidence of the findings from individual studies and to identify those study elements that may help to explain heterogeneity across the body of literature., (Copyright © 2016. Published by Elsevier Ltd.)

Environmental health hazard assessments are routinely relied upon for public health decision-making. The evidence base used in these assessments is typically developed from a collection of diverse sources of information of varying quality. It is critical that literature-based evaluations consider the credibility of individual studies used to reach conclusions through consistent, transparent and accepted methods. Systematic review procedures address study credibility by assessing internal validity or "risk of bias" - the assessment of whether the design and conduct of a study compromised the credibility of the link between exposure/intervention and outcome. This paper describes the commonalities and differences in risk-of-bias methods developed or used by five groups that conduct or provide methodological input for performing environmental health hazard assessments: the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) Working Group, the Navigation Guide, the National Toxicology Program's (NTP) Office of Health Assessment and Translation (OHAT) and Office of the Report on Carcinogens (ORoC), and the Integrated Risk Information System of the U.S. Environmental Protection Agency (EPA-IRIS). Each of these groups have been developing and applying rigorous assessment methods for integrating across a heterogeneous collection of human and animal studies to inform conclusions on potential environmental health hazards. There is substantial consistency across the groups in the consideration of risk-of-bias issues or "domains" for assessing observational human studies. There is a similar overlap in terms of domains addressed for animal studies; however, the groups differ in the relative emphasis placed on different aspects of risk of bias. Future directions for the continued harmonization and improvement of these methods are also discussed., (Published by Elsevier Ltd.)

Objectives: There are some common occupational agents and exposure circumstances for which evidence of carcinogenicity is substantial but not yet conclusive for humans. Our objectives were to identify research gaps and needs for 20 agents prioritized for review based on evidence of widespread human exposures and potential carcinogenicity in animals or humans., Data Sources: For each chemical agent (or category of agents), a systematic review was conducted of new data published since the most recent pertinent International Agency for Research on Cancer (IARC) Monograph meeting on that agent., Data Extraction: Reviewers were charged with identifying data gaps and general and specific approaches to address them, focusing on research that would be important in resolving classification uncertainties. An expert meeting brought reviewers together to discuss each agent and the identified data gaps and approaches., Data Synthesis: Several overarching issues were identified that pertained to multiple agents; these included the importance of recognizing that carcinogenic agents can act through multiple toxicity pathways and mechanisms, including epigenetic mechanisms, oxidative stress, and immuno- and hormonal modulation., Conclusions: Studies in occupational populations provide important opportunities to understand the mechanisms through which exogenous agents cause cancer and intervene to prevent human exposure and/or prevent or detect cancer among those already exposed. Scientific developments are likely to increase the challenges and complexities of carcinogen testing and evaluation in the future, and epidemiologic studies will be particularly critical to inform carcinogen classification and risk assessment processes.

Discovered in the early 1800s, the use of cadmium and various cadmium salts started to become industrially important near the close of the 19th century, rapidly thereafter began to flourish, yet has diminished more recently. Most cadmium used in the United States is a byproduct from the smelting of zinc, lead, or copper ores, and is used to manufacture batteries. Carcinogenic activity of cadmium was discovered first in animals and only subsequently in humans. Cadmium and cadmium compounds have been classified as known human carcinogens by the International Agency for Research on Cancer and the National Toxicology Program based on epidemiologic studies showing a causal association with lung cancer, and possibly prostate cancer, and studies in experimental animals, demonstrating that cadmium causes tumors at multiple tissue sites, by various routes of exposure, and in several species and strains. Epidemiologic studies published since these evaluations suggest that cadmium is also associated with cancers of the breast, kidney, pancreas, and urinary bladder. The basic metal cationic portion of cadmium is responsible for both toxic and carcinogenic activity, and the mechanism of carcinogenicity appears to be multifactorial. Available information about the carcinogenicity of cadmium and cadmium compounds is reviewed, evaluated, and discussed.

Family history is a risk factor for breast cancer and could be due to shared environmental factors or polymorphisms of cancer susceptibility genes. Deficient function of DNA repair enzymes may partially explain familial risk as polymorphisms of DNA repair genes have been associated, although inconsistently, with breast cancer. This population based case-control study examined the association between polymorphisms in XPD (Lys751Gln) and XRCC1 (Arg399Gln and Arg194Trp) genes, and breast cancer. Breast cancer cases (n=321) and controls (n=321) were matched on age and menopausal status. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). The analysis was conducted omitting observations with missing data, and by using imputation methods to handle missing data. No significant association was observed between the XPD 751Gln/Lys (OR 1.37, 95% CI 0.96-1.96) and Gln/Gln genotypes (OR 1.08, 95% CI 0.62-1.86) (referent Lys/Lys), XRCC1 399Arg/Gln (OR 1.48, 95% CI 0.92-2.38) and Gln/Gln genotypes (1.11, 95% CI 0.67-1.83) (referent Arg/Arg) or the XRCC1 Arg/Trp and Trp/Trp genotypes (OR 1.12, 95% CI 0.69-1.83) (referent Arg/Arg) and breast cancer. In multivariate analysis, the adjusted odds ratios for the XPD and XRCC1 399 polymorphisms increased and became statistically significant, however, were attenuated when imputation methods were used to handle missing data. There was no interaction with family history. These results indicate that these polymorphisms in XPD and XRCC1 genes are only weakly associated with breast cancer. Without imputation methods for handling missing data, a statistically significant association was observed between the genotypes and breast cancer, illustrating the potential for bias in studies that inadequately handle missing data.

Glutathione S-transferases (GSTs) are a family of isoenzymes that play an important role in protecting cells from cytotoxic and carcinogenic agents. GSTpi is encoded by the GSTP1 gene. GSTP1 null mice show an increased risk of skin tumorigenesis induced by carcinogens. GSTP1 is transcriptionally silenced by promoter hypermethylation in several human cancers including hepatocellular carcinoma (HCC). Methylation-specific PCR (MSP) was used to analyze the GSTP1 promoter hypermethylation status of 83 hepatocellular carcinoma tissues from Taiwan. Hypermethylation was detected in 38 of 83 (46%) tumors. GSTP1 expression by immunohistochemical staining of HCC tissue samples was significantly associated with methylation status. The relationship between methylation status and clinical parameters and tumor markers including environmental exposure to aflatoxin B1(AFB1) and polycyclic aromatic hydrocarbons (PAH), measured as DNA adducts, was also investigated. A statistically significant association was found between GSTP1 promoter hypermethylation and the level of AFB1-DNA adducts in tumor tissue (OR 2.81, 95% CI 1.03-7.70); a marginally significant association was found for adjacent non-tumor tissue (OR 2.57, 95% CI 0.97-6.80). There was no association between GSTP1 hypermethylation and PAH-DNA adducts in tumor or adjacent non-tumor tissues. These results suggest that epigenetic inactivation of GSTP1 plays an important role in the development of HCC and exposure to environmental carcinogens may be related to altered methylation of genes involved in hepatocarcinogenesis. The mechanism by which environmental exposures induce epigenetic changes in HCC needs further analysis.

O(6)-methylguanine-DNA methyltransferase (MGMT) is a repair protein that specifically removes promutagenic alkyl groups from the O(6) position of guanine in DNA. MGMT is transcriptionally silenced by promoter hypermethylation in several human cancers. Methylation-specific PCR (MSP) was used to analyze the MGMT promoter methylation status of 83 hepatocellular carcinomas (HCC) and 2 HCC cell lines (HepG2 and Hep3B). Hypermethylation was detected in 32 of 83 (39%) HCC tissues, but it was not found in either HCC cell line. We also analyzed MGMT expression by immunohistochemical analysis of HCC tissue samples. The presence of aberrant hypermethylation was associated with loss of MGMT protein. The relationship between methylation status and risk factors and tumor markers including environmental exposure to aflatoxin B(1) (AFB(1)), measured as DNA adducts, and status of tumor suppressor gene p53 was also investigated. A statistically significant association was found between MGMT promoter hypermethylation and high level of AFB(1)-DNA adducts in tumor tissues (OR = 5.05, 95% CI = 1.29-19.73). A significant association was also found between methylation and p53 mutation status (OR = 2.97, 95% CI = 1.09-8.11). These results suggest that epigenetic inactivation of MGMT plays an important role in the development of HCC and exposure to environmental carcinogens may be related to altered methylation of genes involved in cancer development. The role of chemical carcinogens in hypermethylation needs further investigation., (Copyright 2002 Wiley-Liss, Inc.)

Epigenetic changes in gene expression due to extensive CpG island methylation is now accepted as the main cause of inactivation of the p16 gene. More recently, it has been suggested that the human ras association domain family (RASSF) 1 gene, cloned from the lung tumor-suppressor locus 3p21.3, also may be inactivated by methylation. It consists of two major alternative transcripts, RASSF1A and RASSF1C. Epigenetic inactivation of isoform A was observed in several carcinomas and tumor cell lines. In this study, promoter hypermethylation of RASSF1A and p16 was investigated in 83 hepatocellular carcinoma (HCC) tissue samples from Taiwan and in two HCC cell lines (Hep3B and HepG2). High frequencies (85% and 47%, respectively) of methylation of the CpG island promoters of RASSF1A and p16 were found in the HCC tissues. The methylation of RASSF1A also was detected in Hep3B cells but not in HepG2 cells; p16 was not methylated in either cell line. Methylation status was determined in 12 normal control liver tissues and 10 adjacent nontumor tissues. No methylation was found in normal liver control tissues for both RASSF1A and p16; methylation was detected in one of 10 and seven of 10 adjacent nontumor tissue sampless for p16 and RASSF1A, respectively, in subjects with positive tumors. These data indicate that aberrant methylation of the CpG island promoters of both genes is a frequent occurrence in hepatocarcinogenesis. The high frequency of RASSF1A methylation in adjacent tissues suggests that this may be an early event. The relationship between methylation status and clinical parameters and tumor markers, including DNA damage resulting from aflatoxin B(1) (AFB(1)), an environmental carcinogen, and p53 status, also was analyzed. A statistically significant association was found between RASSF1A methylation status and the level of AFB(1)-DNA adducts in tumor tissues. No association was found between methylation status and p53 status. These results suggest the hypothesis that exposure to environmental carcinogens may be involved in altered methylation of genes involved in cancer development., (Copyright 2002 Wiley-Liss, Inc.)

DNA repair efficiency varies among individuals, with reduced repair capacity as a risk factor for various cancers. This variability could be partly explained by allelic variants for different DNA repair genes. We examined the role of a common polymorphism in the XRCC3 gene (codon 241: threonine to methionine change) and bladder cancer risk. This gene plays a role in the homologous recombination pathway, which repairs double-strand breaks. The functional consequences of the XRCC3 codon 241 polymorphism are still unknown. We hypothesized that this polymorphism could affect repair of smoking-associated DNA damage and could thereby affect bladder cancer risk. We genotyped 233 bladder cancer cases and 209 controls who had been frequency matched to cases on age, sex, and ethnicity. We observed little evidence of a positive association between subjects who carried at least one copy of the codon 241 Met allele and bladder cancer (odds ratio: 1.3; 95% confidence interval: 0.9-1.9). Among heavy smokers, individuals with the Met allele had about twice the risk of those without it; however, a test of interaction was not statistically significant (P = 0.26). Previously, we observed in these subjects an association between bladder cancer risk and allelic variants of the XRCC1 gene, which is involved in the repair of base damage and single-strand breaks. In this study, we found some evidence for a gene-gene interaction between the XRCC1 codon 194 and XRCC3 codon 241 polymorphisms (P = 0.09) and some support for a possible gene-gene-smoking three-way interaction (P = 0.08).

HCC is a common cancer and HBV and AFB(1) are well-documented, major risk factors. Epidemiologic studies have documented that cigarette smoking also contributes to the development of HCC. PAHs are ubiquitous environmental pollutants and products of incomplete combustion. They are present in both mainstream and sidestream cigarette smoke. PAHs are metabolically activated by phase I enzymes, including CYP1A1, into electrophilic reactants (diol epoxides), which covalently bind to DNA to form adducts. Diol epoxides are also substrates for phase II detoxifying enzymes, including GSTM and GSTP. To examine the association between PAH-DNA adducts and HCC, adduct levels were determined in liver tissue by relative staining intensity with an immunoperoxidase method using a polyclonal antiserum against BPDE-modified DNA. Subjects were also genotyped for polymorphism in several genes involved in the metabolism of PAH, including GSTM1 and GSTP1. Liver tissue was collected from patients with histologically confirmed HCC (n = 105) and from non-HCC controls (n = 37). There was a significant positive correlation (r = 0.3, p < 0.01) between adducts in tumor and adjacent nontumor tissues among HCC cases. The risk of HCC was higher after adjustment for age, sex and HBsAg in the group with the highest tertile tissue levels of PAH-DNA adducts (mean relative nuclear staining intensity of tumor and nontumor tissue > 344) than in the group with the lowest tertile (staining < 241, OR = 3.9, 95% CI = 1.0-14.9). Among non-HCC controls, there were no significant associations between adduct levels and cigarette smoking, GSTM1 null genotype and HBsAg positivity. A strikingly increased HCC risk was observed (OR = 20.3, 95% CI = 5.0-81.8) among HBsAg-positive subjects whose PAH-DNA adduct levels were high (mean relative nuclear staining intensity of tumor and nontumor tissue > 301, median of control tissues) compared to HBsAg-negative subjects who had low PAH-DNA adduct levels. 4-ABP- and AFB(1)-DNA adducts had been measured previously in these same tissues. Subjects with elevated DNA adduct levels of PAH, 4-ABP and AFB(1) had a significantly higher HCC risk with an OR of 36.7 (95% CI 7.2-187.2) compared to those who had low DNA adduct levels. These results suggest that PAHs may play a role in human hepatocarcinogenesis in conjunction with HBsAg carrier status, GSTM1 and GSTP1 genotypes and exposure to 4-ABP and AFB(1)., (Copyright 2002 Wiley-Liss, Inc.)

X-ray repair cross complementing group 1 (XRCC1) encodes a protein involved in base excision repair. We examined the association of polymorphisms in XRCC1 (codon 194 Arg-->Trp and codon 399 Arg-->Gln) and breast cancer in the Carolina Breast Cancer Study, a population-based case-control study in North Carolina. No association was observed between XRCC1 codon 194 genotype and breast cancer, and odds ratios (ORs) were not modified by smoking or radiation exposure. A positive association for XRCC1 codon 399 Arg/Gln or Gln/Gln genotypes compared with Arg/Arg was found among African Americans (253 cases, 266 controls; OR = 1.7, 95% confidence interval, 1.1-2.4) but not whites (386 cases, 381 controls; OR =1.0, 95% confidence interval, 0.8-1.4). Among African-American women, ORs for the duration of smoking were elevated among women with XRCC1 codon 399 Arg/Arg genotype (trend test; P < 0.001) but not Arg/Gln or Gln/Gln (P = 0.23). There was no difference in OR for smoking according to XRCC1 codon 399 genotype in white women. ORs for occupational exposure to ionizing radiation were stronger for African-American and white women with codon 399 Arg/Arg genotype. High-dose radiation to the chest was more strongly associated with breast cancer among white women with XRCC1 codon 399 Arg/Arg genotype. Our results suggest that XRRC1 codon 399 genotype may influence breast cancer risk, perhaps by modifying the effects of environmental exposures. However, interpretation of our results is limited by incomplete knowledge regarding the biological function of XRCC1 alleles.

Bladder cancer is the sixth most common cancer in the United States. The main identified risk factor is cigarette smoking, which is estimated to contribute to up to 50% of new cases in men and 20% in women. Besides containing other carcinogens, cigarette smoke is a rich source of reactive oxygen species (ROS) that can induce a variety of DNA damage, some of which is repaired by the base excision repair (BER) pathway. The XRCC1 gene protein plays an important role in BER by serving as a scaffold for other repair enzymes and by recognizing single-strand DNA breaks. Three polymorphisms that induce amino acid changes have been found in codon 194 (exon 6), codon 280 (exon 9), and codon 399 (exon 10) of this gene. We tested whether polymorphisms in XRCC1 were associated with bladder cancer risk and whether this association was modified by cigarette smoking. Therefore, we genotyped for the three polymorphisms in 235 bladder cancer cases and 213 controls who had been frequency matched to cases on age, sex, and ethnicity. We found no evidence of an association between the codon 280 variant and bladder cancer risk [odds ratio (OR), 1.2; 95% confidence interval (CI), 0.6-2.6]. We found some evidence of a protective effect for subjects that carried at least one copy of the codon 194 variant allele relative to those homozygous for the common allele (OR, 0.59; 95% CI, 0.3-1.0). The combined analysis with smoking history suggested a possible gene-exposure interaction; however, the results were not statistically significant. Similarly, for the codon 399 polymorphism, our data suggested a protective effect of the homozygous variant genotype relative to carriers of either one or two copies of the common allele (OR, 0.70; 95% CI, 0.4-1.3), and provided limited evidence, albeit not statistically significant, for a gene-smoking interaction.

XPD codes for a DNA helicase involved in transcription and nucleotide excision repair. Rare XPD mutations diminish nucleotide excision repair resulting in hypersensitivity to UV light and increased risk of skin cancer. Several polymorphisms in this gene have been identified but their impact on DNA repair is not known. We compared XPD genotypes at codons 312 and 751 with DNA repair proficiency in 31 women. XPD genotypes were measured by PCR-RFLP. DNA repair proficiency was assessed using a cytogenetic assay that detects X-ray induced chromatid aberrations (breaks and gaps). Chromatid aberrations were scored per 100 metaphase cells following incubation at 37 degrees C (1.5 h after irradiation) to allow for repair of DNA damage. Individuals with the Lys/Lys codon 751 XPD genotype had a higher number of chromatid aberrations (132/100 metaphase cells) than those having a 751Gln allele (34/100 metaphase cells). Individuals having greater than 60 chromatid breaks plus gaps were categorized as having sub-optimal repair. Possessing a Lys/Lys751 genotype increased the risk of sub-optimal DNA repair (odds ratio = 7.2, 95% confidence interval = 1.01-87.7). The Asp312Asn XPD polymorphism did not appear to affect DNA repair proficiency. These results suggest that the Lys751 (common) allele may alter the XPD protein product resulting in sub-optimal repair of X-ray-induced DNA damage.

Cytochrome P450 enzymes play a major role in the metabolism of several of the chemical carcinogens involved in the development of hepatocellular carcinoma (HCC). To investigate by immunohistochemistry interindividual differences in these enzymes, polyclonal antisera and immunoperoxidase staining were used to detect the expression of CYP1A1/2 and 3A4 in 37 surgical control tissues and 105 tumour and adjacent nontumour tissues of HCC cases from Taiwan. There was variability in the expression and staining pattern for both CYP1A1/2 and 3A4 in all tissue types. In tissues from controls, there was no correlation between P450 expression and smoking history or hepatitis B virus antigen status. Since these samples had been previously analysed for the DNA adducts of aflatoxin B1 (AFB1), a dietary mould contaminant, and 4-aminobiphenyl (4-ABP), a component of cigarette smoke, we also investigated the relationship between P450 levels and DNA adducts. 4-ABP-DNA adducts were higher in tissues with elevated levels of CYP1A1/2 (p = 0.02). Overall there was no relationship between CYP1A1/2 or CYP3A4 and AFB1-DNA adducts in control tissues. Staining intensity for CYP1A1/2 and 3A4 followed the order: tumour tissues < control tissues < adjacent non-tumour tissues. CYP1A1/2 levels tended to be lower in tumour and adjacent non-tumour tissues than for CYP3A4. In HCC cases, 4-ABP-DNA adducts were higher in subjects with higher levels of CYP1A1/2, stratified by tissue type, but these differences were not significant. For CYP3A4, in contrast to control tissues, there was a significant association with AFB1-DNA adducts in tumour and adjacent non-tumour tissue of HCC cases. These results suggest that one factor influencing carcinogen-DNA adducts is levels of specific P450 enzymes. However, adduct formation in vivo is a complex processes dependent upon numerous genetic and environmental factors.

Prostate cancer is the most common malignancy in males and is the second most common cause of cancer mortality in American men. Polymorphisms have been identified in two genes, the 17-hydroxylase cytochrome P450 gene (CYP17) and the steroid 5-reductase type II gene (SRD5A2) that are involved with androgen biosynthesis and metabolism. The CYP17 A2 allele contains a T-->C transition in the 5' promoter region that creates an additional Sp1-type (CCACC box) promoter site. The SRD5A2 valine to leucine (V89L) polymorphism has been correlated with lower dihydroxytestosterone levels. We tested genotypes in 108 prostate cases and 167 controls along with samples (n = 340) from several different ethnic groups. The CYP17 A2 allele (combined A1/A2 and A2/A2 genotypes) occurred at a higher frequency in Caucasian patients with prostate cancer (70%) than in Caucasian clinical control urology patients (57%), suggesting that the A2 allele may convey increased risk for prostate cancer [odds ratio (OR) = 1.7, 95% confidence interval (CI) = 1.0-3.0]. Blacks and Caucasians had a similar frequency of the A2 genotype (16 and 17%, respectively) while Taiwanese had the highest frequency (27%). The SRD5A2 leucine genotype was most frequent in Taiwanese (28%), intermediate in Caucasians (8.5%) and lowest in Blacks (2.5%). Genotypes having a SRD5A2 leucine allele were somewhat more common in prostate cancer cases (56%) than in controls (49%) (OR = 1.4, 95% CI = 0.8-2.2) but this difference was not significant. These results support the hypothesis that some allelic variants of genes involved in androgen biosynthesis and metabolism may be associated with prostate cancer risk.

Hereditary genetic defects in DNA repair lead to increased risk of cancer. Polymorphisms in several DNA repair genes have been identified; however, the impact on repair phenotype has not been elucidated. We explored the relationship between polymorphisms in the DNA repair enzyme, XRCC1 (codons 194, 280, and 399), and genotoxic end points measured in two populations: (a) placental aflatoxin B1 DNA (AFB1-DNA) adducts in a group of Taiwanese maternity subjects (n = 120); and (b) somatic glycophorin A (GPA) variants in erythrocytes from a group of North Carolina smokers and nonsmokers (n = 59). AFB1-DNA adducts were measured by ELISA, and erythrocyte GPA variant frequency (NN and NO) was assessed in MN heterozygotes with a flow cytometric assay. XRCC1 genotypes were identified by PCR-RFLPs. The XRCC1 399Gln allele was significantly associated with higher levels of both AFB1-DNA adducts and GPA NN mutations. Individuals with the 399Gln allele were at risk for detectable adducts (odds ratio, 2.4; 95% confidence interval, 1.1-5.4; P = 0.03). GPA NN variant frequency was significantly higher in 399Gln homozygotes (19.6 x 10(-6)) than in Gln/Arg heterozygotes (11.4 x 10(-6); P < 0.05) or Arg/Arg homozygotes (10.1 x 10(-6); P = 0.01). No significant effects were observed for other XRCC1 polymorphisms. These results suggest that the Arg399Gln amino acid change may alter the phenotype of the XRCC1 protein, resulting in deficient DNA repair.

Protein kinase C (PKC) is a central component in signal transduction and growth control and might be an appropriate target for the chemotherapy of human brain tumors. This study demonstrates that the staurosporine derivative Ro 31-8220, a potent PKC inhibitor, inhibited the growth of 7 human brain tumor cell lines with an IC50 of about 2 microM. Calphostin C, a structurally unrelated PKC inhibitor, inhibited the growth of two of these cell lines with an IC50 of about 100 to 300 nM. Drug withdrawal and clonogenicity assays indicated that the growth inhibition by both of these compounds was irreversible. Morphologic studies, DNA fragmentation studies and flow cytometric assays showed that the treated glioblastoma cells underwent apoptosis. Treatment of glioblastoma cells with Ro 31-8220 lead to a rapid decline in the level of the anti-apoptosis protein bcl-2. At least three of the glioblastoma cell lines carried mutant p53 alleles with missense mutations in the DNA binding domain of p53. Therefore, the induction of apoptosis in these cell lines occurred through a p53-independent mechanism. Furthermore treatment of these glioblastoma cell lines with Ro 31-8220 or calphostin C led to an increase of cells in the G2-M phase of the cell cycle. This correlated with a decrease in CDC2-associated histone H1 kinase activity, as well as a decrease in the level of the CDC2 protein as shown by immunoblotting. When added to subcellular assays Ro 31-8220 markedly inhibited CDC2 histone H1 kinase activity with an IC50 of 100 nM, but calphostin C directly inhibited this kinase activity only at very high concentrations (above 100 microM). Thus these compounds inhibit the growth of glioblastoma cells through novel mechanisms. Ro 31-8220, in particular, might be a useful agent for the treatment of human brain tumors.

Recent studies have implicated aflatoxin B1 (AFB1) exposure as an etiological agent in hepatocellular carcinoma (HCC) and suggested an interaction with chronic hepatitis B virus (HBV) infection. Worldwide AFB1 exposure correlates with a specific mutation at codon 249 in the p53 tumor suppressor gene in liver tumors. This study investigated the roles of HBV and AFB1 in the HCC carcinogenic pathway involving p53 mutations. In cases and controls, chronic HBV infection was assessed by serum hepatitis B surface antigen (HBsAg) and AFB1 exposure by immunohistochemical detection of AFB1-DNA adduct in liver tissue. p53 protein mutations in tumor tissues of HCC cases were identified by immunohistochemistry and DNA mutations by single-stranded conformational polymorphism and sequencing analysis. Both chronic HBsAg carrier status and liver AFB1-DNA adducts were significantly higher in cases than in controls with odds ratios (OR) of 8.4 and 3.9, respectively (P < 0.01). Moreover, HCC risk was greatest in individuals with both AFB1-DNA adducts and HBsAg, suggesting a viral-chemical interaction. Mutant p53 protein, mutations in the p53 gene, and specific codon 249 mutations were detected in 37, 29, and 13%, respectively, of the HCC cases. Most of the DNA mutations were transversions, and the only major clustering site for mutations was codon 249. AFB1-DNA adducts were associated with p53 protein (OR = 2.9, P = 0.054) and DNA mutations (OR = 2.9, P = 0.082) but with borderline significance. All of the codon 249 mutations (n = 12) occurred in HBsAg-seropositive carriers, resulting in an OR of 10.0 (P < 0.05), suggesting that HBV may be involved in the selection of these mutations. The ORs between HBsAg and p53 DNA and protein mutations were 2.6 (P = 0.077) and 1.8 (P > 0.05), respectively. Both p53 DNA and protein mutations were related to tumor stage, suggesting that they are late events. These studies provided further support for the role of aflatoxin exposure in HCC in Taiwan and insight into viral-chemical interactions and molecular pathogenesis.

A partial cDNA clone for the interferon (IFN)-induced 67,000-dalton (67K) protein was isolated by immunological screening and used as a probe to study the expression of mRNAs encoding this protein. Northern blot analyses of RNA from IFN-treated GM2767 cells revealed the presence of two major 67K-specific RNA species, 2.7 and 4.3 kb in length, and two minor RNA species, 5.7 and 7.2 kb long. All of these 67K-specific RNAs were polyadenylated. Multiple 67K-specific mRNAs were observed to be induced in several cell lines. IFN-gamma was more effective at inducing these mRNAs than was IFN-alpha. In IFN-alpha-treated GM2767 cells, the 67K-specific mRNAs were detectable 6 h following IFN treatment, but not 12, 18, or 24 h following treatment. In IFN-gamma-treated cells, these mRNAs were detectable 6 h after treatment and continued to be present 24 h after treatment. The induction of the 67K-specific mRNAs in GM2767 cells did not require protein synthesis as the RNAs were induced by IFN-alpha or IFN-gamma in the presence of cycloheximide (CHX, 50 micrograms/ml). Treatment of cells with the combination of CHX and IFN-alpha mediated an enhanced accumulation of the 67K-specific mRNAs, suggesting that ongoing protein synthesis may downregulate the induction or accumulation of the IFN-alpha-induced 67K-specific mRNAs. Western blot analysis employing a monoclonal antibody to the 67K protein revealed that several distinctly sized but immunologically related proteins were induced in IFN-treated cells.

Interferon (IFN) treatment of cells induces the synthesis of several new proteins. Antibody to the IFN-induced 42,000 dalton protein has been prepared and used in the study of this protein. The synthesis of the 42,000 dalton protein is dependent on de novo RNA synthesis, as its induction can be blocked if actinomycin D and the IFN are added to the cells simultaneously. Several lines of evidence suggest that the IFN-induced 42,000 dalton protein is the IFN-induced indoleamine 2,3-dioxygenase (IDO). These are as follows: 1) antibody to the 42,000 dalton protein neutralizes the activity of the IDO; 2) examination of a variety of cell lines reveals a correlation between the presence of this protein and the presence of the IDO; and 3) the induction of both the IDO and the 42,000 dalton protein is blocked under conditions in which the IFN treatment is performed in the presence of cycloheximide, and actinomycin D is added to the cells prior to the removal of the cycloheximide. A study of a variety of cell lines has revealed that the induction of the IDO occurs primarily in response to IFN-gamma. Peripheral blood mononuclear cells (PBMC) were the only cell population in which IFN-alpha and IFN-gamma were observed to produce the IDO. The IDO activity induced in IFN-alpha and IFN-gamma treated PBMC is neutralized by antibody to the 42,000 dalton protein, thus demonstrating that the IDO activity induced in these cells by IFN-alpha and IFN-gamma is mediated by the same molecule or antigenically related molecules. Fractionation of the PBMC populations reveals that it is the monocyte that produces the IFN-induced IDO.

Interferon-gamma (IFN-gamma) treatment of polymorphonuclear leukocytes (PMNs) results in an activation of their functions. Studying the IFN responsiveness of PMNs and using antibodies to the IFN-induced proteins, we have observed the ability of IFN-alpha to stimulate the production of the IFN-induced 67,000 and 56,000 dalton proteins and the ability of IFN-gamma to induce the synthesis of the 67,000, 56,000, and 42,000 dalton proteins. The induction of these proteins is dependent on de novo RNA synthesis, as its induction is inhibited if the IFNs and actinomycin D are added to the cells simultaneously. The results of this study confirm the ability of PMNs to carry out gene activation and demonstrate the ability of PMNs to respond to both IFN-alpha and IFN-gamma.

Interferon (IFN) treatment of cells induces the synthesis of several new proteins. A hybridoma cell line producing monoclonal antibody to the IFN-induced 56,000-dalton protein has been developed. The IFN-induced 56,000-dalton protein is synthesized by a variety of different cells and in response to IFN-alpha, IFN-beta, and IFN-gamma. The induction of this protein is dependent on de novo RNA synthesis, since its induction is inhibited if actinomycin D and the IFNs are added to the cells simultaneously. Labeling of IFN-treated cells at 4-h intervals at various times after the addition of the IFNs reveals that the synthesis of the 56,000-dalton protein in IFN-alpha-treated cells peaks within 12 h after the addition of the IFN and is no longer enhanced 20 h after exposure to the IFN. In contrast, IFN-gamma-treated cells continue to show an enhanced synthesis of this IFN-induced protein even after 20 h of exposure to the IFN. Thus, the synthesis of the IFN-induced 56,000-dalton protein is regulated differently by the different IFNs. When cells are treated with IFN-alpha or IFN-gamma in the presence of cycloheximide, and actinomycin D is added prior to the removal of the cycloheximide, the cells produce the IFN-induced 56,000-dalton protein and develop an antiviral state in response to both IFN-alpha and IFN-gamma. These results demonstrate that the synthesis of the 56,000-dalton protein is not dependent on the synthesis of an intermediary protein and that the establishment of an antiviral state occurs in the absence of multiple transcriptional events.

The treatment of cells with TNF or IFN results in the development of an antiviral state and in the induction of a common set of proteins with m.w. of 80,000, 67,000, and 56,000. The induction of the 80,000- and 56,000-Da proteins after TNF treatment is dependent on the synthesis of an intermediary protein, whereas the induction of the 67,000-Da protein appears to occur as a direct result of the TNF treatment. The effects of antibodies to IFN on the TNF-mediated effects have been evaluated and reveal that the incubation of TNF-treated cells with antibody to rIFN-beta 1 greatly reduces the antiviral effectiveness of the TNF treatment and blocks the ability of TNF to induce the 80,000-Da protein. Incubation with antibodies to either IFN-alpha or IFN-gamma failed to affect the TNF-mediated responses. Thus, the induction by TNF of each of the proteins is regulated differently and is mediated through both IFN-dependent and IFN-independent mechanisms.

Tumor necrosis factor (TNF) or lymphotoxin (LT) treatment of cells sensitive to the anticellular action of TNF results in the degradation of their cellular DNA into fragments that are multiples of about 200 base pairs. The specificity of this DNA fragmenting effect was examined. The DNA of cells dying either as a result of exposure to interferon-gamma (IFN-gamma) or as a result of having exhausted their culture media was observed to be fragmented into multiples of 200 base pairs. Antibody to TNF or LT failed to block the IFN-gamma-mediated DNA fragmentation and antibodies to IFN-gamma, TNF, and LT failed to block the DNA fragmentation observed in the cells dying as a result of having exhausted their culture media. Thus the fragmentation of cellular DNA appears to be nonspecific effect of cell death that can be induced by a variety of treatments.

The treatment of cells sensitive to the anticellular effect of tumor necrosis factor (TNF) with TNF results in a degradation of their cellular DNA into DNA fragments that are multiples of 200 base pairs. TNF treatment of cells resistant to the anticellular effect of TNF, but bearing receptors for TNF, fails to result in any DNA fragmentation. Incubation conditions, such as temperature, the presence of metabolic inhibitors or amino acid deprivation, that modulate the effectiveness of TNF or affect the rate at which TNF exerts its anticellular effect have a similar effect on the ability of the TNF to generate DNA fragments. Thus the TNF-mediated DNA fragmentation and the rate at which it occurs correlates with the rate at which cells respond to the anticellular effect of TNF and, as such, might serve as a marker for the responsiveness of cells to TNF.