Conclusions from VAO and Previous Updates

The committee responsible for the initial VAO report (IOM, 1994) concluded that there was inadequate or insufficient information to determine whether there is an association between exposure to the COIs and respiratory disorders. Additional information available to the committees responsible for Update 1996 (IOM, 1996), Update 1998 (IOM, 1999), Update 2000 (IOM, 2001), Update 2002 (IOM, 2003c), and Update 2004 (IOM, 2005) also maintained the original conclusion of an inadequate or insufficient level of association.

A number of studies of non-malignant respiratory diseases in Vietnam veterans have since been reviewed. However, the majority of these studies were not able to control for major risk factors, such as smoking or tobacco use. Mortality from respiratory diseases was not found to be higher than expected in the Centers for Disease Control and Prevention’s Vietnam Experience Study (Boehmer et al., 2004), in the Air Force Health Study (AFHS) (Ketchum and Michalek, 2005), or in two Australian studies of Vietnam veterans (ADVA, 2005b,c), but it is possible that the use of death certificates may have introduced some misclassification of respiratory deaths (Drummond et al., 2010). In contrast, in the Army Chemical Corps (ACC) cohort of Vietnam veterans, the prevalence of self-reported noncancerous respiratory problems diagnosed by a doctor was significantly increased by about 40–60%, although no differences in the prevalence of respiratory problems were found in the subset of veterans whose serum TCDD was above 2.5 parts per trillion (Kang et al., 2006). Further study of cause-specific mortality in the ACC cohort by Cypel and Kang (2010) found a statistically significant excess of mortality from COPD when comparing the deployed and non-deployed groups. In a later analysis, Cypel and Kang were able to control for self-reported herbicide exposure, body mass index (BMI), and smoking status but found no statistically significant differences in respiratory diseases between sprayers and nonsprayers. Deaths due to COPD were lower in non-deployed ACC veterans than in males in the U.S. population; this is noteworthy because the prevalence of smoking in the non-deployed ACC veterans was about twice that in men in the U.S. population (Kang et al., 2006). Kang et al. (2014a) found lower risks of respiratory system disease deaths and COPD deaths in women veterans who were deployed to Vietnam compared with non-deployed women veterans, but these associations were not statistically significant.

Comparing New Zealand veterans who served in Vietnam with the standardized general male population of New Zealand, the risk of death from all nonmalignant respiratory diseases (excluding COPD) was statistically significantly lower, but for COPD specifically no difference was found (McBride et al., 2013). Researchers of the Korean Veterans Health Study applied the Stellman exposure model and found no statistically significant difference for deaths from respiratory disease (Yi et al., 2014a,b). A comparison of deaths in the low-exposure category with the deaths in the high-exposure category found a statistically significant risk for non-malignant respiratory diseases that was statistically significantly elevated for COPD but not for pneumonia or asthma. A separate analysis of disease prevalence in the Korean Veterans Health Study used insurance claim data and found no difference in the risk of diseases of the respiratory system overall between the high-exposure and the low-exposure groups, but it did report statistically significant differences in the risks for COPD, pneumonia not due to influenza, chronic bronchitis, bronchiectasis, and asthma (Yi et al., 2014a).

Several studies of occupational cohorts have also been reviewed by the many VAO Update committees. Update 2000 (IOM, 2001) drew attention to findings in the Seveso cohort that suggested a higher mortality from non-cancerous respiratory disorders in study subjects, particularly males, who were more heavily exposed to TCDD. Additional follow-up of mortality in the Seveso cohort found some increase in mortality from COPD at the 15- and 20-year points (Bertazzi et al., 1998, 2001) as well as at the 25-year follow-up (Consonni et al., 2008).

Occupational and industrial cohorts in the United States, the United Kingdom, New Zealand, and Australia did not find increased mortality from non-cancerous respiratory diseases overall (Boers et al., 2010; Collins et al., 2009a,c; McBride et al., 2009a), though they were unable to control for smoking. An update of the National Institute for Occupational Safety and Health (NIOSH) cohort of pentochlorophenol (PCP) workers (Ruder and Yin, 2011) did not find an association between exposure to the COIs and deaths from all non-malignant respiratory diseases; no association with COPD deaths was found for the subgroup exposed to both TCDD and PCP. Updated mortality data on workers in two chlorphenoxy herbicide plants in the Netherlands were reanalyzed by Boers et al. (2012) using serum measurements, but no association of TCDD exposure with respiratory diseases was observed. An updated mortality study of workers in a pesticide factory with TCDD contamination (Manuwald et al., 2012) showed no association of non-malignant respiratory disease with exposure.

No associations were observed with respiratory mortality in a small subcohort of New Zealand phenoxy herbicide sprayers ('t Mannetje et al., 2005) or with mortality from COPD in private applicators or their spouses in the Agricultural Health Study (AHS) (Blair et al., 2005a). Several other studies using the AHS cohort that examined morbidity from particular self-reported respiratory health problems, including wheeze, asthma, “farmer’s lung” or hypersensitivity pneumonitis, and chronic bronchitis were examined by update committees, but most findings were not statistically significant.

Based on the many studies considered in various cohorts that applied different designs, measures of exposure, and definitions of non-malignant respiratory outcomes, the conclusion of inadequate or insufficient evidence of an association between exposure to the COIs and mortality from all non-cancerous respiratory diseases or from COPD specifically, has remained unchanged. There also remains inadequate or insufficient evidence of an association between exposure to the COIs and the prevalence of respiratory diseases, such as wheeze or asthma, COPD, and farmer’s lung.

Update of the Epidemiologic Literature

This section summarizes the results of the relevant studies on respiratory disorders and exposures to the COIs. One new study of Vietnam veterans and respiratory disorders, one occupational study, and three other relevant studies of respiratory disorders and the COIs have been identified since Update 2014. Tables 43 and 44, which can be found at www.nap.edu/catalog/25137, summarize the results of studies related to non-cancer respiratory disease and COPD and pulmonary function, respectively.

Biologic Plausibility

Several recent studies explored the pro- and anti-inflammatory activity of TCDD exposure and of aryl hydrocarbon receptor (Ahr) in lungs in mice and in mouse and human lung cells. These studies addressed such health effects as lung response to viral infections, asthma, COPD, and allergen-induced lung inflammation. Cho et al. (2015) found that nuclear coactivator 7 (NCOA7) showed differential activity in a cell-culture study of TCDD exposure in normal lung epithelial and lung cancer cells with NCOA7 increasing CYP1A1 activity and the levels of inflammatory cytokines in the diseased cells but not in normal cells. S. M. Lee et al. (2017) found that AHR activation by the endogenous ligand kynurenine plays a role in immune regulation in inflammatory responses in a mouse model of lung injury.

Q. Liu et al. (2014) exposed juvenile zebrafish to TCDD in diet and reported a novel finding of lesions in nasal epithelium. Boule et al. (2015) exposed mice to TCDD during development and investigated their response to viral infection as adults. Treated mice had increased bronchopulmonary inflammation with an enhanced CD4+ T-cell response.

X. M. Li et al. (2016) found that TCDD-Ahr activation prevented inflammation and airway hyper-responsiveness in a non-eosinophilic asthma model in mice. The lungs of treated mice showed reduced eosinophil and neutrophil infiltration, and Th17 cytokine IL-17 was reduced in serum and bronchio-alveolar lavage fluid (with Th2 cytokine IL-4 also reduced). In a study comparing Ahr knockout and wild-type mice, T. Xu et al. (2015) found that TCDD treatment enhanced mesenchymal stem cell recruitment, thereby suppressing allergen-induced lung inflammation.

Sarill et al. (2015) explored the role of AHR and suggested that AHR has a role regulating anti-oxidant defenses in lung structural cells. In Ahr knockout mouse lung fibroblasts and human lung adenoma cells that were deficient in AHR expression and exposed to cigarette smoke extract, they found increased oxidative stress compared to wild-type cells. They also showed that anti-oxidant gene induction was significantly less in the Ahr knockout mouse lung fibroblasts. In lung fibroblasts from mice with an Ahr mutation (incapable of DNA binding), they showed that anti-oxidant sulfiredoxin 1 induction after cigarette smoke-extract exposure was independent of dioxin-response element binding (i.e., a role for AHR independent of exogenous ligand). In sum, the findings of Sarill et al. (2015) suggest that low AHR expression may facilitate the development or progression of COPD. Zago et al. (2017) studied the role of AHR and Cox-2 protein suppression and also concluded that low AHR contributes to increased inflammation, based on their study in human lung fibroblasts.

Synthesis

Update 2014 included new data that did not add any compelling evidence of an association of exposure to the COIs and respiratory disorders. The current update still does not display a coherent body of epidemiologic evidence to support conclusions concerning whether the risks of other particular respiratory problems are associated with exposure to the COIs. Cox et al. (2015) showed an increase in hospitalization risk rates for COPD and pneumonia, but not asthma, in New Zealand Vietnam veterans. However, smoking was not controlled for in the analysis and exposure to the COIs was not verified. In a 30-year follow-up of workers who produced 2,4,5-T in New Zealand, highly exposed workers did not have an elevated risk of doctor-diagnosed asthma, chronic bronchitis, tuberculosis, pleurisy, or pneumonia compared with lower-exposed workers based on job duties or serum TCDD concentration. The study of electric arc foundry workers in Italy (Cappelletti et al., 2016) reported no difference in the risk of asthma, but this was based on two cases. Moreover, the primary exposure, foundry dust, is a complex mixture, and it is not possible to isolate any potential COI exposure. In a follow-up of TCP and PCP workers at the Dow Midland, Michigan, plant, Collins et al. (2016) did not find elevated mortality for respiratory system disorders. Henneburger et al. (2014) found no association of asthma exacerbation with exposure to the COIs in the AHS. Therefore, the prior assessment cannot be altered since the new findings are mixed, and the study designs have limitations.

Conclusion

On the basis of the evidence reviewed here and in previous VAO reports, the committee concludes that there is inadequate or insufficient evidence of an association between exposure to the COIs and mortality from all non-cancerous respiratory diseases or from COPD specifically. There is also inadequate or insufficient evidence of an association between exposure to the COIs and the prevalence of respiratory disorders.

Peptic-Ulcer Disease

Peptic-ulcer disease refers to ulcerative disorders of the gastrointestinal tract that are caused by the action of acid and pepsin on the stomach or duodenal mucosa. Peptic-ulcer disease is characterized as gastric or duodenal ulcer, depending on the site of origin. Peptic-ulcer disease occurs when the corrosive action of gastric acid and pepsin overcomes the normal mucosal defense mechanisms that protect against ulceration. About 10% of the population has clinical evidence of having had a duodenal ulcer at some time in their lives; a similar percentage is affected by gastric ulcer. The incidence of duodenal ulcer peaks in the fifth decade, and the incidence of gastric ulcer about 10 years later.

Evidence increasingly indicates that the bacterium Helicobacter pylori is linked to peptic-ulcer disease (both duodenal and gastric). H. pylori colonizes the gastric mucosa in 95–100% of patients who have a duodenal ulcer and in 75–80% of patients who have a gastric ulcer. Healthy people in the United States under 30 years old have gastric colonization rates of about 10%. Over the age of 60 years, colonization rates exceed 60%. Colonization alone, however, is not sufficient for the development of ulcer disease; only 15–20% of subjects who have H. pylori colonization will develop ulcers in their lifetime. Other risk factors include genetic predisposition (such as some blood and human leukocyte antigen types), cigarette smoking, and psychologic factors (chronic anxiety and stress).

Liver Disease

Blood tests of liver function are the mainstay of diagnosing liver disease. Increases in serum bilirubin and in the serum concentrations of some hepatic enzymes—aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and γ-glutamyltransferase (GGT)—are commonly noted in liver disorders. The relative sensitivity and specificity of those enzymes for diagnosing liver disease vary, and a diagnosis can require several tests. The only regularly reported abnormality in liver function associated with TCDD exposure in humans is an increase in GGT. The estimated serum activity of that enzyme is a sensitive indicator of a variety of conditions, including alcohol and drug hepatotoxicity, infiltrative lesions of the liver, parenchymal liver disease, and biliary tract obstruction. Increases are noted after many chemical and drug exposures that are not followed by evidence of liver injury. The confounding effects of alcohol use (often associated with increased GGT) make it difficult to interpret changes in GGT in exposed people (Calvert et al., 1992). An increase in GGT can be considered a normal biologic adaptation to chemical, drug, or hormone exposure.

Cirrhosis is the most commonly reported liver disease in epidemiologic studies of herbicide or TCDD exposure. Cirrhosis is an irreversible chronic injury of the liver with extensive scarring and a resulting loss of function. Clinical symptoms and signs include jaundice, edema, abnormalities in blood clotting, and metabolic disturbances. Cirrhosis can lead to portal hypertension with associated gastroesophageal varices, an enlarged spleen, abdominal swelling attributable to ascites, and, ultimately, hepatic encephalopathy that can progress to coma. It is generally impossible to distinguish the various causes of cirrhosis through the clinical signs and symptoms or pathologic characteristics. The most common cause of cirrhosis in North America and many parts of western Europe and South America is excessive alcohol consumption. Other causes are chronic viral infection (hepatitis B or hepatitis C), the poorly understood condition primary biliary cirrhosis, chronic right-sided heart failure, and a variety of less common metabolic and drug-related conditions.

Conclusions from VAO and Previous Updates

Several studies reviewed by previous VAO committees have reported on several non-cancerous digestive system outcomes, and for that reason this section and the update of new epidemiologic literature presents all results for digestive and hepatobiliary outcomes by publication. Some studies that have been reviewed focused on liver enzymes, while others reported on specific liver diseases. An evaluation of the effects of herbicide and TCDD exposure on non-cancerous gastrointestinal ailments is challenging in that clinical experience suggests that medical history and physical examination are undependable diagnostic tools for some ailments, so incidence data are sometimes problematic. The strong interdependence among the characteristics of a given person (such as weight and the laboratory indexes of hepatic function and health) and TCDD body burden complicates the already difficult task of assessing association.

Studies of Vietnam veterans have generally examined different laboratory endpoints and clinical conditions, making comparisons and overall conclusions difficult. An analysis of the AFHS found that a significantly higher percentage of Ranch Hand veterans in the high-dioxin category had excesses of transaminase and other nonspecific laboratory measures of liver function than their comparison subjects. The data were consistent with an interpretation of a dose–response relationship, but other explanations were also plausible (AFHS, 2000). Later analyses also reported some abnormalities in liver enzymes among the Ranch Hands, including decreasing levels of C4 complement as dioxin increased and abnormal triglyceride concentrations that increased as the 1987 dioxin concentration increased (AFHS, 2005). However, mortality studies of the Ranch Hand cohort have not found an increased mortality related to gastrointestinal or liver disease (Ketchum and Michalek, 2005). Among ACC veterans, an increased rate of hepatitis was associated with Vietnam service but not with a history of spraying herbicide (Kang et al., 2006), and an 80% excess of digestive system or cirrhosis deaths was observed in veterans who handled or sprayed herbicides in Vietnam compared with their non-Vietnam-veteran peers (Cypel and Kang, 2010).

Studies of digestive and liver disease among other Vietnam veterans cohorts have not found associations with presumed herbicide exposure. The Australian Vietnam-veterans study (ADVA, 2005b) did not find an increase in liver disease in military personnel who served in Vietnam compared with the general population of Australia. The relationship between possible herbicide exposure and liver and gastric-ulcer disease was described in a sample of Korean Vietnam-era veterans in three publications by Yi et al. (2013, 2014a,b). Based on health insurance claims data, the adjusted prevalence of peptic-ulcer disease was found to be 3% higher in those with high exposure than in those with low exposure after adjusting for several behavioral, demographic, and service-related factors. For liver cirrhosis, there was likewise a small elevation in the prevalence and a significant log-linear relationship between an exposure opportunity score and the odds of having cirrhosis. In the mortality study in the same cohort of Korean Vietnam veterans, there was no association between putative log-transformed exposure and mortality from peptic ulcers (Yi et al., 2014b). However, highly exposed veterans were 17% more likely to die from cirrhosis than those with low exposure. Deaths from alcoholic liver disease were also statistically significantly elevated in the more highly exposed veterans.

Most of the analyses of occupational or environmental cohorts have had insufficient numbers of cases to support confident conclusions. Gastric ulcer was not associated with serum levels of dioxin-like PCDD/Fs or dioxin-like PCBs or with total TEQs after adjusting for possible confounders in a general population sample of 2,264 Japanese adults (Nakamoto et al., 2013). An analysis of National Health and Nutrition Examination Survey (NHANES) data on blood levels of 37 environmental pollutants and alanine aminotransferase levels in 1,345 persons aged 12 years and older found that, in general, blood levels of dioxin-like chemicals were not significantly correlated with alanine aminotransferase levels. The exception was 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, which was associated with only a slight elevation (< 1%) (Yorita Christensen et al., 2013).

A study of the IARC cohort of phenoxy-herbicide and chlorophenol production workers and sprayers (Vena et al., 1998), the only study that had a relatively large number of observations, found less digestive system disease and cirrhosis mortality in exposed workers than in non-exposed controls. The mortality results through 2001 for the Seveso, Italy, cohort (Consonni et al., 2008) found no excess of deaths related to digestive diseases or specifically to cirrhosis. Several mortality studies of various occupational cohorts exposed to the COIs have been inconsistent but generally found no statistically significant increases in deaths from either ulcers or cirrhosis (Boers et al., 2010, 2012; Collins et al., 2010a,b; Manuwald et al., 2012; McBride et al., 2009a,b; Ruder and Yiin, 2011).

Thus, the reports have been inconsistent, and interpreting individual studies is difficult because of a lack of information on alcohol consumption and other risk factors. In the studies that showed the strongest association between potential exposure and gastrointestinal disease (specifically cirrhosis), there was strong evidence that excess alcohol consumption was the cause of the cirrhosis. The committee responsible for VAO concluded that there was inadequate or insufficient information to determine whether there is an association between exposure to the COIs and gastrointestinal and digestive disease, including liver toxicity. Additional information available to the committees responsible for subsequent updates has not changed that conclusion.

Update of the Epidemiologic Literature

One new study of gastrointestinal diseases among Vietnam veterans and two occupational studies of workers in herbicide-producing plants have been identified since Update 2014.

Biologic Plausibility

The liver is the first organ that encounters chemicals absorbed from the gastrointestinal tract, and it is responsible for metabolizing them to water-soluble chemicals that can be excreted in the urine. Because the liver has many detoxifying enzymes that efficiently metabolize many chemicals, liver toxicity is usually associated only with high-dose acute exposure or lower-dose chronic exposure. The liver can be damaged if the metabolism of a chemical results in the production of a reactive intermediate that is more toxic than the parent chemical. Changes in the serum concentrations of liver enzymes are biomarkers of liver toxicity, and their magnitudes correlate with the degree of liver damage. The exposure of laboratory animals to high doses of 2,4-D, 2,4,5-T, and TCDD is known to cause liver damage. The mechanisms by which the phenoxy herbicides damage the liver are based on the inhibition of mitochondrial function by the blocking of oxidative phosphorylation; this leads to a loss of generation of adenosine triphosphate, the death of cells, and hepatic necrosis and fibrosis. TCDD-induced hepatotoxicity is mediated by the activation of the AHR, which leads to changes in gene transcription and associated changes in cell function. Changes in gene expression are associated with several physiologic processes, oxidative stress, and apoptosis (Boverhof et al., 2005, 2006). TCDD-mediated hepatic steatosis is characterized by the accumulation of triglycerides caused by the combined up-regulation of CD36/fatty acid translocase and fatty acid transport proteins, the suppression of fatty acid oxidation, the inhibition of the hepatic export of triglycerides, an increase in peripheral fat mobilization, and an increase in hepatic oxidative stress (J. H. Lee et al., 2010). Recent evidence suggests that hepatic steatosis produced by TCDD might be mediated by the mitochondria (He et al., 2013). The exposure of rats to TCDD over a 2-year period (NTP, 2004) also produced several changes in the liver, including hepatocyte hypertrophy, multinucleated hepatocytes, inflammation, pigmentation, diffuse fatty change, necrosis, bile duct hyperplasia, bile duct cyst, nodular hyperplasia, portal fibrosis, and cholangiofibrosis. Lamb et al. (2016) studied a mouse model of liver injury and found that TCDD exposure increased liver damage and inflammation, suggesting that TCDD-activated AHR enhanced hepatic stellate cell activity.

AHR displays species differences; for example, amino acid sequences in the C-terminal region of human and mouse AHR are only 58% identical. Compared with the mouse Ahr, the human AHR has about a 10-fold lower relative affinity for TCDD; the difference has been attributed to the amino acid residue valine 381 in the ligand-binding domain of the human AHR (Flaveny et al., 2009; Ramadoss and Perdew, 2004). The existence of species differences associated with AHR activation is supported by the divergence in the transcriptomic and metabolomic responses to TCDD in mouse, rat, and human liver (Boutros et al., 2008, 2009; Carlson et al., 2009; Forgacs et al., 2012, 2013; Kim et al., 2009; Nault et al., 2013; Watson et al., 2017). In a recent study, gene-expression changes were compared in adult female primary human and rat hepatocytes that had been exposed to TCDD in vitro (Black et al., 2012). Whole-genome microarrays found that TCDD produced divergent gene-expression profiles in rat and human hepatocytes, both on an ortholog basis (conserved genes in different species) and on a pathway basis. For commonly affected orthologs or signaling pathways, the human hepatocytes were about 15-fold less sensitive than rat hepatocytes. Another microarray study examining species-specific transcriptomic differences in primary hepatocytes from humans, mice, and rats identified 16 orthologous genes that were dysregulated by TCDD in all three species (Forgacs et al., 2013). Such findings are consistent with epidemiologic studies that have shown humans to be less sensitive to TCDD-induced hepatotoxicity. However, it should be noted that in vitro human hepatocyte studies may not reflect the in vivo response of human liver to TCDD.

There have been few reports of health-relevant effects of phenoxy herbicides or TCDD on the gastrointestinal tract, even after high exposure. A series of recent papers demonstrated the influence of TCDD and the Ahr pathway on the development of the gut microbiome in mice (Stedtfeld et al., 2017a,b,c), which is thought to influence immune response that may affect gastrointestinal disorders. However, the available animal data do not support a plausible link between herbicide exposure and gastrointestinal toxicity in Vietnam veterans.

Synthesis

Previous updates indicated that there have been inconsistent findings for the COIs and gastrointestinal and liver disease and that interpreting individual studies is difficult because of a lack of information on alcohol consumption (especially for liver cirrhosis) and other risk factors. The recent studies of Vietnam veterans from New Zealand (Cox et al., 2015) and occupational cohorts of phenoxy herbicide producers (Collins et al., 2016; 't Mannetje et al., 2018) have not provided informative evidence for an association with the COIs. Although a few animal studies have shown an influence of TCDD and the Ahr pathway on the development of the gut microbiome in mice (Stedtfeld et al., 2017a,b,c), which may modify the immune response in gastrointestinal disorders. The available animal data do not support a plausible link between herbicide exposure and gastrointestinal toxicity in Vietnam veterans.

Conclusion

On the basis of the evidence reviewed here and in previous VAO reports, the committee concludes that there is inadequate or insufficient evidence to determine whether there is an association between exposure to the COIs and gastrointestinal and digestive diseases.

Conclusions from VAO and Previous Updates

Publications from the Korean Veterans Health Study included findings for non-malignant kidney disease. Yi et al. (2013a) examined the prevalence of self-reported exposure based on six questions and self-reported kidney failure using a postal survey of 114,562 Korean veterans who had served in the Vietnam War. The incidence of kidney failure was compared by defining high and low categories of exposure based on the six exposure questions and also by exposure opportunity index (EOI) scores that were calculated. Self-reported kidney failure was statistically significantly increased when the analysis was based on perceived exposures, but it was not significant when the analysis was based on the EOI scores. In a study of cause-specific mortality through 2005 for 180,639 Korean veterans who were alive in 1992, Yi et al. (2014b) found that after adjustment for age in 1992 and rank, no differences in the hazard ratios were observed for acute renal failure or for chronic renal failure when the analyses were based on the EOI scores.

Two environmental studies of non-U.S. populations were also considered. In a cross-sectional study of 2,264 Japanese men and women who had not been occupationally exposed to dioxins, self-reported kidney disease (not otherwise specified) was not associated with serum levels of dioxin-like PCDD/Fs or dioxin-like PCBs or with total TEQs after adjusting for possible confounders (Nakamoto et al., 2013). The second study sought to determine the factors contributing to a form of kidney disease not related to diabetes, hypertension, or any other recognized cause in adults in Sri Lanka (Jayatilake et al., 2013). Urine samples were taken from 57 cases and from 39 controls who were from non-endemic areas, and the samples were analyzed for 11 biomarkers of pesticides, including the COIs 2,4-D, 2,4,5-T, and 2,4,5-TCP. Of these, only 2,4-D was among the seven biomarkers found at concentrations above the limit of detection; 3.5% of the cases had 2,4-D concentrations above the reference limit of 0.3 μg/L. Since the urinary pesticide results were presented for only the cases, no inference can be made about the relative risk for this kidney condition in association with 2,4-D.

Based on these four studies, the committee for Update 2014 concluded that there was inadequate or insufficient evidence of exposure to the COIs and nonmalignant kidney diseases.

Update of the Epidemiologic Literature

One new study of Vietnam veterans and kidney and urinary disorders has been identified since Update 2014, as well as supporting occupational, environmental, and case-control studies of kidney and urinary disorders and exposure to the COIs.

Biologic Plausibility

Studies of mice, rats, goldfish, and zebrafish have documented kidney toxicity from TCDD exposure. Studies of 2,4-D in goldfish and TCDD in rats report oxidative stress in kidneys (Matviishyn et al., 2014; Palaniswamy et al., 2014). Q. Liu et al. (2014) investigated the effects of developmental exposure to TCDD in zebrafish and found kidney lesions and the dysregulation of the genes involved in renal necrosis and cell death as well as decreased hematopoietic cells in the kidney marrow. Aida-Yasuoka et al. (2014) reported a chance finding that C57BL/6J mouse pups are more susceptible to TCDD-induced hydronephrosis than BALB/cA mice; in pups exposed to TCDD on postnatal days 1–7, the prevalence of hydronephrosis was 64% in the C57BL/6J pups and 0% in BALB/cA pups. In both strains of mice, the Ahr receptors are highly responsive to TCDD; however, genetic differences were found in expression of renal m-Prostaglandin E synthase-1 and early growth response 1 (Egr-1). In a study of mice, Bu et al. (2017) found that Ahr activation up-regulates glucose transporter 9 (Glut9), which plays a role in maintaining uric acid homeostasis.

Synthesis

Since Update 2014, seven studies have been reviewed for kidney disease and urinary disorder outcomes related to exposure to the COIs. A hospitalization study of New Zealand Vietnam veterans found that chronic renal failure risk was statistically significantly increased among the veterans compared with the standardized general population of New Zealand, but there was no difference in the prevalence of other kidney or urinary outcomes (Cox et al., 2015); however, there was no exposure validation, some of the conditions (such as urinary tract infections) do not typically require hospitalization, and potentially important risk factors were not adjusted for in the analysis. In a 30-year follow-up study of New Zealand workers in a plant that produced 2,4,5-T, having a doctor-diagnosed kidney function problem was not different for workers in high- versus low-exposure groups, based on reported job and duties in the plant or on serum TCDD levels. Two analyses of end-stage renal disease in the AHS (Lebov et al., 2015, 2016) found no statistically significant associations with the phenoxy herbicides 2,4-D or 2,4,5-T among the male pesticide applicators or their wives. In an analysis of NHANES, Everett and Thompson (2016) evaluated the association of the blood levels of three chlorinated dibenzo-p-dioxins, one chlorinated dibenzofuran, and four dioxin-like PCBs with nephropathy among 1,505 adolescents and young adults and found that dioxin-like chemicals were associated with nephropathy among young females, but not males, although the effect estimates were very imprecise. An environmental exposure study of Taiwanese residents living in close proximity to a former PCP-producing factory found that those who had high serum dioxin levels had a statistically significantly elevated risk of chronic kidney disease (Huang et al., 2016). A second analysis of this population was identified (Chang et al., 2013), but because it examined hyperuricemia, which is a measure of disturbed metabolism and not a recognized health outcome, it was not considered as part of the evidence base of chronic kidney conditions related to exposure to the COIs. A cross-sectional study of agricultural behaviors, including the use of 2,4-D, and health outcomes in a Nicaraguan community (Raines et al., 2014) found a decreased glomerular filtration rate to be associated with cutting sugarcane, sugarcane chewing, and non-deliberate pesticide inhalation, but no serum or other objective measure of exposure were collected. Studies of mice, rats, goldfish, and zebrafish document kidney toxicity from TCDD exposure, but such outcomes do not present a consistent mechanism for the kidney dysfunction diseases in humans. Epidemiology studies concerning exposure to the COIs and kidney diseases were not reported prior to Update 2014. However, the new epidemiologic findings reviewed do not present a coherent pattern of an association between exposure to the COIs and kidney or urinary disorders.

Conclusion

After reviewing the new evidence of exposure to the COIs and non-malignant kidney and urinary outcomes, the committee concludes that there remains inadequate or insufficient evidence of an association between exposure to the COIs and non-malignant kidney or urinary disorders.

Conclusions from VAO and Previous Updates

Thyroid homeostasis in humans was first addressed with respect to the COIs by the committee for Update 2002. After consideration of several new studies and because of the consistent observations of exposures to the COIs being related to perturbations of thyroid function—and to clinical hypothyroidism in particular—the committee for Update 2014 considered the body of epidemiologic data, in combination with strong biologic plausibility, to represent limited or suggestive evidence of an association between exposure to the COIs and hypothyroidism. Additional endocrine effects have been observed in conjunction with exposure to the COIs in both humans and animals, but the evidence is inadequate or insufficient to establish an association with herbicide exposure for them.

An extensive assessment of endocrine function in clinical examinations, including a series of thyroid-function tests, failed to show systematic differences in thyroid function between Ranch Hands and comparison veterans (AFHS, 1991a). In analyzing individual TCDD readings obtained for subjects in the AFHS, however, Pavuk et al. (2003) found statistically significantly increased TSH measures from the 1985 and 1987 examinations in the high-exposure category and a significant increasing trend across the three TCDD categories during the 1982, 1985, 1987, and 1992 examinations. No increased risk of disorders of the thyroid was found among a study of Australian Vietnam veterans (O’Toole et al., 2009). Among Korean Vietnam veterans, two publications considered thyroid outcomes (Yi et al., 2014a,b). The first (Yi et al., 2014a) used claims data to report on the prevalence of disorders of the thyroid gland in 111,726 veterans and found an increased risk of thyroid conditions overall (ICD-10 E00–E07) with herbicide exposure, after adjustment for several factors. The pattern was very similar for both non-iodine-deficiency hypothyroidism (ICD-10 E03) and for other nontoxic goiter (ICD-10 E04). The risk of thyroiditis (ICD-10 E06) overall was not found to be significantly associated with herbicide exposure, but the strongest endocrine-related results were for autoimmune thyroiditis (ICD-10 E06.3), a subcategory of thyroiditis. No difference in the risk of hyperthyroidism (ICD-10 E05) was found between high- and low-exposure groups. Comparing the high-exposure group with the low-exposure group based on EOI scores, an elevated risk was observed for pituitary hypofunction (p = 0.011), while the risk of pituitary hyperfunction was not different between high- and low-exposure groups. The risk of hyperaldosteronism was also not elevated. A mortality analysis of the Korean Vietnam veteran cohort of 180,639 male veterans did not find any association between herbicide exposure and deaths from endocrine diseases when the cohort was analyzed as a group (Yi et al., 2014b).

Few studies of thyroid function have been conducted among occupational cohorts exposed to the COIs. Calvert et al. (1999) provided evidence of higher adjusted mean free-T4 concentrations in TCDD-exposed workers in the NIOSH Cross-Sectional Medical Study, but there was no dose–response relationship with serum TCDD. Bloom et al. (2006) found indications of an inverse relationship between the sum of dioxin-like chemicals and the concentration of free T4 in anglers in New York State, but no association between the sum of dioxin-like chemicals and TSH or T3. An analysis of the AHS that was restricted to male private pesticide applicators examined self-reported physician-diagnosed hyperthyroidism, hypothyroidism, and other thyroid conditions and the use of 50 specific agents, including 2,4-D, 2,4,5-T, and 2-(2,4,5-triphenoxy) proprionic acid (2,4,5-TP) (Goldner et al., 2013). After adjusting for age, education, and BMI, the odds of hypothyroidism for ever-use versus never-use were significantly elevated for 2,4-D, for 2,4,5-T, and for 2,4,5-TP. In comparison with those who had never used 2,4-D, an increased risk of hypothyroidism was seen in both those who had used 2,4-D for more than the median number of days and those whose days of 2,4-D use were fewer than the median (p-trend = 0.025). The use of 2,4,5-TP was found to be associated with a decreased risk of hyperthyroidism. None of the phenoxy herbicides were found to be related to having histories of other thyroid diseases. In an Italian study that compared urban and rural workers, Ciarrocca et al. (2012) found that the workers’ urinary arsenic levels differed by a factor of between 2 and 4, and urinary arsenic was positively correlated with serum TSH and thyroglobulin and negatively with free T3 and T4.

The results from several environmental studies have also been reviewed by update committees, and no evidence was found of effects on thyroid function or disease in women exposed to pesticides (Chevrier et al., 2008) or, among women in the AHS, exposed to phenoxy herbicides (Goldner et al., 2010). Using data from NHANES III (1988–1994), Schreinemachers (2010) did not find associations between recent exposure to 2,4-D and T4 or TSH concentrations. An analysis of 1999–2002 NHANES data (Turyk et al., 2007) found total T4 to have a weak inverse relationship with serum TEQs; the effect was somewhat stronger in people over 60 years old and in women.

Among women exposed in the Seveso incident, a significant inverse association was found between serum concentrations of TCDD over a 20-year period (1976–1996) with serum total T4, but not with TSH or free T3, which were measured in 1996. This association was stronger for women who were exposed before menarche than for women exposed after menarche. When thyroid hormones were measured again in 2008 and compared with TCDD levels in 1976 and 1996, the association was no longer present (Chevrier et al., 2014). In a cross-sectional study of 2,264 Japanese men and women who had not been occupationally exposed to dioxins, self-reported thyroid disease (not otherwise specified) was not associated with serum levels of dioxin-like PCDD/Fs or dioxin-like PCBs or with total TEQs after adjusting for possible confounders (Nakamoto et al., 2013). Abdelouahab et al. (2008) described thyroid function in adult freshwater-fish consumers in Canada; dioxin-like congeners were associated with an increase in TSH and a decrease in T4 but below the threshold at which clinical symptoms would be present. Clear effects of dioxin-like chemicals on thyroid function were not apparent in Inuit adults (Dallaire et al., 2009) or in a cross-sectional study of a Chinese community exposed to an electronic-waste recycling plant (J. Zhang et al., 2010). Manh et al. (2013) and Kido et al. (2014) studied steroid hormone levels in the serum and saliva and dioxin concentrations in the breast milk of lactating Vietnamese women living in an “Agent Orange hot spot” (n = 51) or in an area with no suspected exposure (n = 58). Levels of cortisol and corticosterone in serum and saliva were higher in those women living in the hot spot area and were positively correlated with breast-milk dioxin concentrations. Trnovec et al. (2013) measured thyroid gland volume and free T4 in 320 adults from an organochlorine-contaminated area in Slovakia. Blood samples from these subjects produced readings above the limits of detection for all 7 dioxins, 8 of 10 furans, and all but 1 of the 12 PCBs on the 2005 WHO list of dioxin-like chemicals.

Update of the Epidemiologic Literature

One new epidemiological study of occupational exposures to the COIs and thyroid or endocrine effects has been identified for this update. Table 45, which can be found at www.nap.edu/catalog/25137, summarizes the results of studies related to thyroid homeostasis that have been reviewed in the VAO series.

Biologic Plausibility

The influence of TCDD on thyroid-hormone homeostasis has been measured in numerous animal studies, and TCDD exposure has been associated with changes in serum concentrations of T4, T3, and TSH. In most studies, TCDD exposure is associated with a hypothyroid state, including reduced circulating T3 and T4 and increased TSH, especially after chronic exposure. The reduction in circulating T4 concentrations is robust and has recently been proposed as a biomarker of the effect of dioxin-like chemicals (J. M. Yang et al., 2010). Female rats exposed chronically to TCDD showed follicular-cell hyperplasia and hypertrophy of thyroid follicles, which were consistent with an overstimulation of the thyroid by TSH (TSH increases as a homeostatic response to low T4 levels) (Yoshizawa et al., 2010). TCDD enhances the metabolism of thyroid hormones primarily through an AHR-dependent induction of glucuronyl transferase activity (Gessner et al., 2012; Y. Kato et al., 2010; Martin et al., 2012; Nishimura et al., 2005). An enhanced accumulation of T4 in hepatic tissue of TCDD-treated mice may also contribute to the reduction in circulating T4 (Y. Kato et al., 2010).

The possibility that arsenic could act as an endocrine disruptor on thyroid hormone–mediated processes has been proposed on the basis of cell-culture studies and experiments with the ex vivo amphibian tail metamorphosis assay (Davey et al., 2008). In guinea pigs that were fed diets containing 50 ppm arsenic as sodium arsenite or arsenic trioxide for 11 weeks, serum (total) T3 and T4 were reduced compared to controls by about 20–25% and 33%, respectively (Mohanta et al., 2014). Recent data in zebrafish show that arsenite exposure increases thyroxine levels and alters the expression of genes in the hypothalamic–pituitary–thyroid axis, including thyroid receptors α and β, TSH, and corticotropin-releasing hormone (H. J. Sun et al., 2015). These data raise the possibility that cacodylic acid may also disrupt thyroid homeostasis, but there are no published epidemiologic studies that have addressed this.

Synthesis

Numerous animal experiments and several epidemiologic studies have shown that TCDD and dioxin-like chemicals exert some influence on thyroid homeostasis, with the findings being most consistently indicative of hypothyroidism (Boas et al., 2006, 2012; Chevrier et al., 2014). The underlying molecular mechanisms resulting in these effects on thyroid hormone and TSH concentrations in humans, however, are not yet fully characterized. In addition, there are some data to suggest the possibility that arsenic-based herbicides may also affect thyroid function.

Several previous studies of populations environmentally exposed to PCBs found some combination of elevated TSH concentrations and depressed T4 and T3 levels (Bloom et al., 2006; Hagmar et al., 2001a; Persky et al., 2001; Schell et al., 2004), although some (Hagmar et al., 2001b; Sala et al., 2001) found no significant effect. For example, Pavuk et al. (2003) reported a trend of higher TCDD serum concentrations being associated with increasing concentrations of TSH that was not accompanied by changes in circulating T4 or T3 (which would be interpreted as subclinical hypothyroidism) in participants in the AFHS. This finding in U.S. Vietnam veterans is complemented by the results from the Korean Veterans Health Study (Yi et al., 2014a), which found evidence of an increased occurrence of clinical hypothyroid disease, possibly associated with autoimmune hypothyroidism, in association with higher estimated potential herbicide exposure. In addition, the report from the AHS of increased physician-diagnosed hypothyroidism in herbicide applicators with phenoxy herbicide exposure (Goldner et al., 2013) supports the notion that this association is real. Results from the Korean Veterans Health Study suggest that adrenal and possibly pituitary function may also be affected by exposure to dioxin-like chemicals.

For the current update, in vitro and animal data continue to support previous findings that the COIs can alter thyroid homeostasis. There is additional new data (Sun et al., 2015) that raise the possibility that cacodylic acid might similarly disrupt the normal action of the thyroid. There is little additional epidemiologic data available regarding the association of the COIs with thyroid disease or other endocrine function. A follow-up of phenoxy herbicide producers in New Zealand did not find any difference in thyroid disorders between high- and low-exposure groups ('t Mannetje et al., 2018).

Conclusion

On the basis of the new evidence and that reviewed in previous VAO reports, the committee concludes that there is limited or suggestive evidence of an association between exposure to at least one of the COIs and hypothyroidism. There is inadequate or insufficient evidence for disruption of thyroid homeostasis or other endocrine disorders.

Conclusions from VAO and Previous Updates

The committee responsible for VAO (IOM, 1994) determined that there was sufficient evidence of an association between exposure to at least one COI (TCDD) and chloracne. Additional information available to the committees responsible for Update 1996 (IOM, 1996), Update 1998 (IOM, 1999), Update 2000 (IOM, 2001), Update 2002 (IOM, 2003c), Update 2004 (IOM, 2005), and Update 2006 (IOM, 2007) maintained that conclusion. Even in the absence of a full understanding of the cellular and molecular mechanisms that lead to the disease, several notable reviews (Panteleyev and Bickers, 2006; Sweeney and Mocarelli, 2000) have deemed the clinical and epidemiologic evidence of dioxin-induced chloracne to be strong. The occupational epidemiologic literature has many examples of chloracne in workers after reported industrial exposures (Beck et al., 1989; Bond et al., 1987, 1989a,b; Cook et al., 1980; Goldman, 1972; May, 1973, 1982; Oliver, 1975; Pazderova-Vejlupkova et al., 1981; Poland et al., 1971; Suskind and Hertzberg, 1984; Suskind et al., 1953; Zober et al., 1990). With relative risk estimates as high as 5.5 in exposed workers compared with referent non-exposed workers, Bond et al. (1989a) identified a dose–response relationship between probable exposure to TCDD and chloracne. Not everyone who is exposed to relatively high doses develops chloracne, and some with lower exposure may demonstrate the condition (Beck et al., 1989).

Almost 200 cases of chloracne were recorded among those residing in the vicinity of the accidental industrial release of dioxin in Seveso, Italy; most cases were in children and in those who lived in the highest-exposure zone, and most had resolved within 7 years (Assennato et al., 1989a,b; Caramaschi et al., 1981; Mocarelli et al., 1991).

Exposures of Vietnam veterans were substantially lower than those observed in occupational studies and in environmental disasters, such as in Seveso. The long period since the putative exposure has imposed methodologic limitations on the studies of Vietnam cohorts for chloracne. Nonetheless, the Vietnam Experience Study (CDC, 1988a) found that chloracne-like lesions were self-reported more often by Vietnam veterans than by Vietnam-era veterans (OR = 1.4; 95% CI 0.7–2.9). No excess risk was found in Vietnam versus era veterans among subjects who had dermatologic examinations (OR = 0.9, 95% CI 0.7–1.2 for comedones and acne from lesions; OR = 1.2, 95% CI 0.9–1.7 for hyperpigmentation). Compared with matched, non-exposed controls, Ranch Hand personnel reported a significant excess of acne (OR = 1.6, 95% CI 1.1–2.1) (Wolfe et al., 1990), but no cases of chloracne or post-inflammatory scars were found on physical examination 20 years after the potential herbicide exposure (AFHS, 1991b).

Update of Epidemiologic Literature

Three new studies of exposure to the COIs and chronic skin conditions have been identified since Update 2014. However, each study examined different outcomes, making comparisons among the studies difficult.

Biologic Plausibility

Previous updates have reported that chloracne-like skin lesions have been observed in several animal species in response to exposure to TCDD but not to purified phenoxy herbicides. Data accruing over the past several decades demonstrated that TCDD alters the differentiation of human keratinocytes, and more recent studies have illuminated how. TCDD accelerates the events associated with early differentiation and also obstructs the completion of differentiation (Bock, 2017a,b; Fontao et al., 2017; Geusau et al., 2005; Mandavia, 2015). Panteleyev and Bickers (2006) proposed that the major mechanism of TCDD induction of chloracne is activation of the stem cells in the basal layer of the skin to differentiate and then an inhibition of their ability to commit fully to a differentiated status. De Abrew et al. (2014) elucidated the role of matrix metalloproteinase-10 in the histopathological changes that typify chloracne. Work by Tauchi et al. (2005) and K. J. Smith et al. (2017) implicated additional inflammation-related mechanisms by which TCDD exposure may lead to chloracne. The data provide biologically plausible mechanisms for the induction of chloracne by TCDD.

Synthesis

No epidemiologic data in the past decade have refuted the conclusion of prior VAO committees that the evidence of an association between exposure to dioxin and chloracne is sufficient. Because TCDD-associated chloracne becomes evident within a few months after exposure, there is no risk of new cases long after service in Vietnam. Given the established relationship of an association between TCDD and chloracne and the long period that has elapsed since service in Vietnam, the present committee concludes that the emergence of persistent additional biologic or epidemiologic evidence that would merit review and deliberation by later VAO committees is unlikely. The formation of chloracne lesions and other skin disorders after the administration of TCDD has been observed in some species of laboratory animals. The newly identified epidemiologic studies (Cox et al., 2015; Mitoma et al., 2015; 't Mannetje et al., 2018) examined different outcomes and had varying exposure specificity, thereby limiting the comparisons that could be made. Although some differences between exposed and unexposed populations were noted, each study had several limitations that did not change the evidence of association with exposure to the COIs and chronic skin conditions.

Conclusion

On the basis of numerous epidemiologic studies of occupationally and environmentally exposed populations and supportive toxicologic information, previous VAO committees have consistently concluded that there is sufficient evidence of an association between exposure to at least one COI and chloracne. After review of new literature, the committee concludes that there is inadequate or insufficient evidence of an association between exposure to the COIs and other chronic skin conditions.

Conclusions from VAO and Previous Updates

Update 2010 considered one study of Australian Vietnam veterans that found that the veterans had a higher prevalence of all the eye conditions assessed—cataracts, presbyopia, color blindness, and other diseases of the eye—than the Australian population (O’Toole et al., 2009). However, the committee noted a lack of information on exposure to the COIs and a lack of clinical confirmation of the eye conditions, and it had serious concerns about the possibility that recall bias had played a role in the findings. On the basis of that one study, the committee for Update 2010 concluded that there was inadequate or insufficient evidence to determine whether there is an association between exposure to the COIs and eye conditions. No new epidemiologic studies of exposure to the COIs and eye problems had been published for review in Update 2012 and Update 2014.

Update of Epidemiologic Literature

Only one new study of eye conditions was identified. Cox et al. (2015) used hospital discharge records from 1988 to 2009 to identify prevalent health conditions in 2,783 male New Zealand veterans who had served in Vietnam during 1964–1972. Age-specific hospitalization rates were calculated using the total number of annual hospitalizations published by the Ministry of Health and the average annual resident population. Standardized hospitalization rates and 99% CIs were calculated for the veteran cohort and the general population, and an SHR was calculated using the two rates for two conditions related to the eyes. Neither the risk of hospitalization for cataracts (n = 99; SHR = 1.11, 99% CI 0.82–1.40) nor retinal disease (n = 31; SHR = 1.35, 99% CI 0.73–1.98) was different between the veterans and general New Zealand population. Cataract and retinal disease are not generally conditions that require hospitalization, and therefore, the estimated prevalence may be higher. Exposure was not validated through serum measurements and was assumed based on deployment to Vietnam.

Biologic Plausibility

There have been several recent reports of ocular activity associated with Ahr activation or TCDD exposure of rats (Sugamo et al., 2009), mice (Takeuchi et al., 2009), and human non-pigmented ciliary epithelial cells (Volotinen et al., 2009). Hu et al. (2013) reported that mice harboring the null allele at the Ahr locus developed macular age-related degeneration-like pathology.

Woeller et al. (2016) investigated whether AHR activation had therapeutic potential for thyroid eye disease, which includes myofibroblast accumulation and orbital scarring. TGF-β induces myofibroblast formation, and AHR influences TGF-β signaling. Using orbital fibroblasts cultured from thyroid eye disease patients, Woeller et al. found that exposure to AHR ligands 6-formylindolo(3,2-b)carbazole2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (FICZ and ITE) prevented TGF-β-induced myofibroblast formation.

Synthesis

Hospital discharge data from New Zealand Vietnam veterans (Cox et al., 2015) do not suggest any association between exposure to the COIs and retinal disease or cataracts. The data generated in vitro or in vivo in animal models reveal a quite modest possibility of ocular activity for the COIs; these are limited data that do not assist the committee in determining an association between exposure to the COIs and eye conditions.

Conclusion

Given the lack of additional evidence, the committee concludes that there is inadequate or insufficient evidence to determine whether there is an association between exposure to the COIs and eye conditions.

Conclusions from VAO and Previous Updates

Update 2010 was the first report in the VAO series to review studies of the association between exposures to the COIs and a decrease in bone mineral density. Few studies have reported results in this category with enough exposure specificity to be fully informative. Hodgson et al. (2008) studied the relationship between environmental exposures and bone mineral density in a set of 325 members of the Osteoporosis Cadmium as a Risk Factor (OSCAR) cohort who were at least 60 years old. Forearm bone mineral density was measured, blood samples were analyzed for the five dioxin-like mono-ortho PCB congeners (PCB 105, 118, 156, 157, and 167), and TEQs were calculated. In men, PCB 118 was significantly negatively associated with bone mineral density, but there was no association between the TEQ for any of the five dioxin-like mono-ortho PCBs and bone mineral density. In women, PCB 118 and the TEQs for all five dioxin-like mono-ortho PCBs were positively associated with bone mineral density. When the risk of low bone mineral density was treated as a binary variable in an adjusted logistic model, there was a significant association with PCB 118 in men, but none of the measured compounds was predictive in women. A study of 350 women who were exposed to TCDD as a result of the chemical explosion in Seveso, Italy, examined the relationship of DEXA-assessed bone mineral density and TCDD serum levels (Eskenazi et al., 2014). The results suggested that TCDD levels were associated with some evidence of better bone structure in the 48 women for whom exposure occurred after peak bone mass, which is estimated to happen 2 years after menarche, but the findings did not support the hypothesis that postnatal TCDD exposure adversely affects adult bone health. Two cross-sectional studies that examined the association of exposure to dioxin-like chemicals and bone quality in residents of Canada’s northern regions, who are known to be exposed to these chemicals as a result of their diet of marine mammals and predatory fish (Paunescu et al., 2013a,b), found an increase in dioxin-like PCBs 105 and 108 to be negatively associated with a “stiffness index,” and neither total plasma dioxin-like chemicals levels nor any specific dioxin-like PCB level was associated with ultrasonography-assessed bone strength in Inuit women.

Update of the Epidemiologic Literature

Only one new study of bone conditions was identified. Cox et al. (2015) used hospital discharge records from 1988 to 2009 to identify health conditions that had affected 2,783 male New Zealand veterans who had served in Vietnam during 1964 to 1972. Age-specific hospitalization rates were calculated using the total number of annual hospitalizations published by the Ministry of Health and the average annual resident population. Standardized hospitalization rates and 99% CIs were calculated for the veteran cohort and the general population, and an SHR was calculated using the two rates for conditions related to other joint diseases and other connective tissue diseases. An elevated risk of hospitalization for other joint diseases (n = 77; SHR = 1.34, 99% CI 1.04–1.64) and other connective tissue disease (n = 169; SHR = 1.25, 99% CI 1.06–1.44), neither of which is further defined, was found for veterans when compared with the standardized New Zealand population. Since these disease categories were not clearly defined, it is difficult to interpret the findings. The study is also limited because the exposures were not validated through serum measurements and were assumed based on deployment to Vietnam.

Biologic Plausibility

Animal studies suggest that TCDD may have some influence on bone formation and maintenance. Recent work from Herlin et al. (2013) showed that the exposure of adult mice to TCDD resulted in harder bone matrix, thinner cortical bone, mechanically weaker bones, and, most notably, increased trabecular bone volume fraction in Ahr knockout mice. It is known that TCDD can induce chondrocyte apoptosis in culture, which could be an initial event leading to the sort of cartilage degradation observed in arthritis (Yang and Lee, 2010). Lee and Yang (2012) recently demonstrated that this is mediated by reactive oxygen species. In addition, TCDD exposure via the dam’s milk impaired bone mineralization during postnatal development in mice because of a reduction in osteoblastic activity as a result of TCDD-induced up-regulation in the active form of vitamin D in serum (Nishimura et al., 2009). TCDD altered osteogenesis (bone formation) in an in vitro osteoblast model and led to alterations in the proteins associated with cytoskeleton organization and biogenesis, a decrease in the expression of calcium-binding proteins, and a decrease in osteoblast calcium deposition (Carpi et al., 2009). In adult rats, TCDD exposure reduced trabecular bone cross-sectional area, but it significantly increased total bone mineral density; it was further noted that TCDD decreased the expression of the bone-formation marker procollagen type I N-terminal propeptide and increased the expression of the bone-resorption marker carboxy-terminal collagen cross-link, suggesting a net loss of bone tissue (Lind et al., 2009). It is also known that exposure to polyaromatic hydrocarbons (such as those in tobacco smoke) can affect bone health, and some of these alterations have been shown to be mediated, at least in part, by the AHR. That implies that TCDD may alter or modify the effects of osteoblast formation and function as well as inhibit osteoclast formation and function (Kung et al., 2012; Yan et al., 2011). Iqbal et al. (2013) recently addressed this, studying genetically altered mice in order to understand the contributions of tobacco carcinogens and TCDD. In their work, mice in which the Ahr or CYP1A1, CYP1A2, and CYP1B1 genes were deleted displayed reduced resorption and high bone mass. In contrast, Ahr activation by administering benzo[a]pyrene to wild-type mice increased osteoclastogenesis and bone resorption.

Synthesis

The small amount of available epidemiologic information on the possible adverse effects of exposure to the COIs on bone structure has previously been based entirely on dioxin-like mono-ortho PCBs, which contribute a small percentage to total TEQs based on all dioxin-like PCBs. The recent data from New Zealand Vietnam veterans, while suggesting a slightly statistically elevated risk of “other” bone and joint conditions, has poorly described outcomes (with essentially no data on the exact bone conditions considered) and is further limited by the use of hospitalization rates for such outcomes, which are difficult to interpret. Biological data confirm that TCDD is active in bone metabolism, but the pattern of association of exposure to the COIs and subsequent disease is not consistent in the current literature.

Conclusion

There is inadequate or insufficient evidence of an association between exposure to the COIs and clinical or overt adverse effects of osteoporosis, loss of bone mineral density, or other bone conditions.