Free Radicals Several investigators have suggested that oxidative stress plays a critical role in TCDD toxicity (Enan and Matsumura, 1995). Although the contribution of the AhR to the oxidative stress response elicited by TCDD continues to be debated, recent evidence suggests that the response is mediated by the AhR complex (Alsharif et al., 1994). In some studies, congenic mice differing at the Ah locus were administered TCDD (5-125 µg/kg) as a single oral dose, and these studies showed dose-dependent increases in superoxide anion production by macrophages from the TCDD-responsive C57BL/6J (bb) mice relative to control cells. Only the highest dose of TCDD produced a significant increase in superoxide anion formation in C57BL/6J (dd) mice. The acute toxic effects of TCDD may involve TNFα, a cytokine which sensitizes and activates phagocytic cells to agents that elicit release of reactive oxygen species (Alsharif et al., 1994). TNFα may act as an amplifying loop in TCDD-induced oxidative stress, as suggested by the ability of anti-TNFα to reduce oxidative stress.

Protein Kinases The interaction of AhR signaling with protein kinase C (PKC)-dependent signal transduction has received the most attention during the past two years (Carrier et al., 1992; Okino et al., 1992; Berghard et al., 1993; Weber et al., 1994). The interaction of proteins with DNA with each other, or with other transcription factors is influenced at the post-transcriptional level. For instance, the AhR is phosphorylated on both serine and threonine residues (Perdew, 1992), and dephosphorylation of the ligand/AhR complex by phosphatase acid decreases the DNA binding ability of both cytosolic and nuclear AhR preparations (Pongratz et al., 1991). Mahon and Gasiewicz (1995) have recently confirmed the suggestion that the AhR itself is a phosphoprotein and further demonstrated that phosphorylation sites are also present in the C-terminal half of the protein, a region within or adjacent to a DNA-binding repressor domain. In their study, total AhR phosphorylation was not altered by transformation, but phosphorylation was implicated in the formation of the active complex that associates with cis-acting regulatory elements. The DNA-binding ability of the dephosphorylated AhR can be restored by addition of a PKC inhibitor-sensitive cytosolic factor (Berghard et al., 1993). Co-administration of TCDD and TPA to C57BL/6J mice inhibits the accumulation of TCDD-inducible liver CYP1A1 mRNA, and this effect is paralleled by a reduction in DNA binding of the AhR complex to the XRE secondary to loss of nuclear AhR levels (Okino et al., 1992). In addition, TPA significantly decreases EROD activity in human MCF-7 breast cancer cells (Moore et al., 1993).

Carrier et al. (1992) have shown that 2-aminopurine, an inhibitor of protein kinase activity, inhibits induction of CYP1A1 mRNA and corresponding enzymatic activity by TCDD. In their studies formation of DNA/AhR complexes was also inhibited by this agent. Hepa-1 cells treated with staurosporine prior to induction with TCDD were unable to form active complexes, while depletion of PKC by prolonged treatment with PMA suppressed CYP1A1 induction by TCDD.



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--> Free Radicals Several investigators have suggested that oxidative stress plays a critical role in TCDD toxicity (Enan and Matsumura, 1995). Although the contribution of the AhR to the oxidative stress response elicited by TCDD continues to be debated, recent evidence suggests that the response is mediated by the AhR complex (Alsharif et al., 1994). In some studies, congenic mice differing at the Ah locus were administered TCDD (5-125 µg/kg) as a single oral dose, and these studies showed dose-dependent increases in superoxide anion production by macrophages from the TCDD-responsive C57BL/6J (bb) mice relative to control cells. Only the highest dose of TCDD produced a significant increase in superoxide anion formation in C57BL/6J (dd) mice. The acute toxic effects of TCDD may involve TNFα, a cytokine which sensitizes and activates phagocytic cells to agents that elicit release of reactive oxygen species (Alsharif et al., 1994). TNFα may act as an amplifying loop in TCDD-induced oxidative stress, as suggested by the ability of anti-TNFα to reduce oxidative stress. Protein Kinases The interaction of AhR signaling with protein kinase C (PKC)-dependent signal transduction has received the most attention during the past two years (Carrier et al., 1992; Okino et al., 1992; Berghard et al., 1993; Weber et al., 1994). The interaction of proteins with DNA with each other, or with other transcription factors is influenced at the post-transcriptional level. For instance, the AhR is phosphorylated on both serine and threonine residues (Perdew, 1992), and dephosphorylation of the ligand/AhR complex by phosphatase acid decreases the DNA binding ability of both cytosolic and nuclear AhR preparations (Pongratz et al., 1991). Mahon and Gasiewicz (1995) have recently confirmed the suggestion that the AhR itself is a phosphoprotein and further demonstrated that phosphorylation sites are also present in the C-terminal half of the protein, a region within or adjacent to a DNA-binding repressor domain. In their study, total AhR phosphorylation was not altered by transformation, but phosphorylation was implicated in the formation of the active complex that associates with cis-acting regulatory elements. The DNA-binding ability of the dephosphorylated AhR can be restored by addition of a PKC inhibitor-sensitive cytosolic factor (Berghard et al., 1993). Co-administration of TCDD and TPA to C57BL/6J mice inhibits the accumulation of TCDD-inducible liver CYP1A1 mRNA, and this effect is paralleled by a reduction in DNA binding of the AhR complex to the XRE secondary to loss of nuclear AhR levels (Okino et al., 1992). In addition, TPA significantly decreases EROD activity in human MCF-7 breast cancer cells (Moore et al., 1993). Carrier et al. (1992) have shown that 2-aminopurine, an inhibitor of protein kinase activity, inhibits induction of CYP1A1 mRNA and corresponding enzymatic activity by TCDD. In their studies formation of DNA/AhR complexes was also inhibited by this agent. Hepa-1 cells treated with staurosporine prior to induction with TCDD were unable to form active complexes, while depletion of PKC by prolonged treatment with PMA suppressed CYP1A1 induction by TCDD.

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--> These data were interpreted to suggest that phosphorylation is necessary for the formation of a transcriptional complex and for activation of the CYP1A1 gene. In agreement with this view, pretreatment of mouse hepatocyte cultures and hepatoma cells with staurosporine inhibited TCDD-activated transactivation of CYP1A1 in a dose-dependent manner (Chun et al., 1994). Tyrphostin AG213, a specific tyrosine kinase inhibitor, had no effects on TCDD-induced CYP expression, further supporting the view that PKC is the kinase involved in this process. However, the effects of TCDD on PKC and inositol phosphate metabolism in primary cultures of rat hepatocytes exhibit markedly different profiles from phorbol esters, suggesting that these tumor promoters modulate hepatocyte signal transduction via distinct mechanisms (Wolfle et al., 1993). Thus, it remains to be determined with certainty whether PKC is involved in phosphorylation of the ligand/AhR complex or whether phosphorylation of the AhR occurs subsequent to ligand binding and transformation. This issue was addressed by Schafer et al. (1993), who examined the role of PKC on the functionality of the hepatic cytosolic Ah receptor. In these experiments, two nonspecific PKC inhibitors, H7 and staurosporine, and one specific PKC inhibitor, calphostin c, were employed in assays of Ah receptor transformation and DNA binding. AhR transformation and DNA binding occurred in hepatic cytosol despite the absence of detectable kinase activity. Functional PKC activity was not required for ligand-dependent transformation or DNA binding of the Ah receptor complex. An interesting finding in their studies was that staurosporine, a nonspecific kinase inhibitor, formed an AhR/DNA complex that co-migrates with that produced by TCDD and can effectively compete with TCDD for DNA binding. Although some controversy remains, it appears that phosphorylation events occur early in the process of AhR complex formation in the absence of ligand binding. Phosphorylation may influence the stability of the AhR protein and/or DNA binding. The biological significance of TCDD-induced changes in protein phosphorylation remains to be defined. TCDD modulates PKC as well as other kinases in somatic cells at low concentrations, and these effects can influence the physiology of the cells. For instance, a dose-dependent increase in tyrosine phosphorylation of five hepatic intracellular proteins has been reported in TCDD-treated C57BL/6J female mice (Ma et al., 1992). TCDD induces a rapid rise in protein phosphorylation activities in the extranuclear fraction (i.e., cytosol and cellular membranes) of the adipose tissue from male guinea pigs (Enan and Matsumura, 1994). This effect occurs both in vivo and in vitro and is not abolished by actinomycin D, an inhibitor of transcription. However, inhibition of protein synthesis by cycloheximide partially suppresses the effect of TCDD. These responses correlate with a quick rise in ras GTP binding activity, as well as phosphorylation of nuclear c-myc protein. In view of the lack of inhibition by actinomycin D and the short time required for TCDD to induce phosphorylation, it has been suggested that stimulation of protein phosphorylation activities by TCDD is not mediated via a transcriptional process. In support of this interpretation

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--> is the finding that TCDD activates protein phosphorylation activity under cell-free conditions and in the absence of nuclear protein. The ability of TCDD to influence protein phosphorylation is seen in cells of varying embryologic origin and at different stages of differentiation. For instance, the ability of TCDD to influence PKC may involve cell-cycle-related events, became TCDD and its structural analogs decrease PKC activity in Go-synchronized cell populations (Weber et al., 1994) but increase activity in cycling cells (Weber et al., 1995b). Exposure of immature thymocytes to TCDD transiently increased detergent-extractable PKC activity. At later time points, PKC activity returned to control values (DePetrillo and Kurl, 1993). TCDD induces an increase in protein-tyrosine kinases in pancreas at early stages of poisoning (Ebner et al., 1993). Most critical to this discussion is the finding that alterations in intracellular protein tyrosine kinase phosphorylation is a more sensitive indicator of TCDD exposure than EROD induction (Ma et al., 1992). Anti-Estrogenicity TCDD has been shown to interact with the estrogen signal transduction pathway. Harper et al. (1994) have recently demonstrated that AhR ligands can inhibit estrogen-induced responses in MCF-7 human breast cancer cells. TCDD inhibited 17β-estradiol (E2)-induced progesterone receptor (PR) binding, immunoreactive protein, nuclear PR formation, and PR mRNA levels. Scatchard analysis of PR binding demonstrated that TCDD decreased the number of E2-induced PR binding sites, but not the binding affinity for promegestrone. For a series of halogenated aromatics including TCDD, 2,3,7,8- and 1,2,7,8-tetrachlorodibenzofuran, 1,3,7,8-TCDD, and 6-methyl-1,3,8-trichlorodibenzofuran, their rank order of potency for inhibiting E2-induced PR binding paralled their rank order for binding to the AhR, suggesting a role for the receptor in the antiestrogenic activity of polynuclear and halogenated aromatic hydrocarbons. The antiestrogenic effects of TCDD on 17β-estradiol-induced pS2 expression has recently been examined in several cell types, including MCF-7, ZR-75, HeLa, and Hepa-1c1c7 wild-type and mutant cells (Zacharewski et al., 1994). The responses to TCDD were compared to a weaker AhR congener, 2,8-dichlorodibenzo-p-dioxin, and to the well-established antiestrogens ICI 164,384 and tamoxifen. Their results implicated a role for the Ah receptor in TCDD-mediated suppression of E2-induced pS2 expression and suggested that antiestrogenicity required sequences within the pS2 promoter other than the estrogen response element. These results are particularly significant became since pS2 has been proposed as a prognostic marker for breast cancer (Koerner et al., 1992). The effects of E2 alone or in combination with TCDD have recently been evaluated in weanling female S-D rats (White et al., 1995). In this species, TCDD did not influence E2-induced responses including uterine weight and keratinization of the vaginal epithelium, suggesting that the antiestrogenic effects of TCDD may exhibit species-, strain-, and age-dependence.

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--> Estimating Potential Health Risk and Factors Influencing Toxicity TEF Approach Toxic equivalency factors (TEFs) have been proposed to estimate the potential health risk associated with exposure to dibenzo-p-dioxins, dibenzofurans, and polyhalogenated biphenyls and to complex mixtures containing these chemicals (Safe, 1990). This approach assumes linearity of the toxic response for TCDD and related chemicals and is based on a receptor-medicated mechanism of action. The TEF approach has come under increasing scrutiny, because the relative inductive potency of these chemicals may be tissue-specific (De Vito et al., 1993) and may be influenced by additive and negative interactions among the chemicals present in complex mixtures (Van den Berg et al., 1994); the approach also disregards potentially significant kinetic interactions. As such, estimates of TEFs based on hepatic ethoxyresorufin-O-deethylase (EROD) may not accurately reflect the potency of these chemicals in nonhepatic tissue or the chronic toxicity of the agents in question. Clearly, additional research is needed to begin to resolve this controversy. Interspecies and Interindividual Differences in Sensitivity Species differences in susceptibility to TCDD toxicity are best exemplified by differences in LD50 values. The large differences in LD50 values between species and strains was previously discussed and depicted in Table 4-1 of Chapter 4 of the NAS 1994 publication of Veterans and Agent Orange. The suggestion has been made that humans are less sensitive to the toxic effects of TCDD than other species employed (Dickson and Buzik, 1993). Studies conducted by Fan and Rozman (1995) demonstrate that Long Evans rats are less susceptible to TCDD toxicity than are Sprague-Dawley rats (Fan and Rozman, 1995). Subtle differences in the regulation of intermediary metabolism between these two rat strains appear to be responsible for strain differences in susceptibility to TCDD. Hepatic phosphoenol pyruvate carboxy kinase (PEPCK) activity is decreased in a dose-dependent manner in Long Evans and in Sprague-Dawley rats, indicating inhibition of gluconeogenesis. This is an early and reversible effect of TCDD (Fan and Rozman, 1995). Inhibition of feed intake appeared to be secondary to elevated serum tryptophan levels. Hepatic gamma-glutamyl transferase was also reduced in a dose-dependent fashion. Dose-dependent responses in Long Evans rats occurred in a higher dose range than those required for the reduction of PEPCK activity, thus providing an explanation for the decreased susceptibility of the Long-Evans rats to TCDD relative to Sprague-Dawley rats. While such pharmacogenetic differences have been defined in laboratory species, we are only beginning to gain an appreciation of such differences in human populations, as evidenced by cytochrome P450 polymorphisms (Landi et al., 1994). The relative toxic potency of four chlorinated dibenzo-p-dioxins (CDDs) in two species with different sensitivities (guinea pig, Sprague-Dawley rat) was reviewed by Rozman et al. (1993). These investigators defined a dissociation between AhR-mediated enzyme induction and acute toxicity in these two species

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--> and suggested that AhR-mediated effects cannot be fundamental to the mechanism of acute toxicities of CDDs. Of perhaps more significance was the finding that the relative toxic potencies for CDDs in rats were similar for acute, subchronic, and chronic doses, suggesting that multiplicity of effects reflects different time-dependent adaptive responses of the organism. Tissue Specificity A recent study by Thomas and Gallo (1994) demonstrated that the polyamines putrescine, spermidine, and spermine induce changes in the sedimentation profile and DNA binding of the AhR. Spermidine and spermine caused the precipitation of the 9S oligomeric receptor, with a gradual decrease in receptor peak during density gradient sedimentation. RNase A treatment transformed the 9S AhR to a 6S form, and DNA binding increased by twofold. Following partial purification of transformed AhR, it lost the ability to bind to DNA, but addition of spermidine increased DNA binding in a concentration-dependent manner, suggesting that polyamines modulate the structure and DNA binding of the AhR. These data raise the intriguing possibility that polyamine levels are important in the tissue-specific toxicity and the cell-cycle-specific effects of TCDD in somatic cells. Significant Interactions Dietary Interactions Significant interactions between TCDD toxicity and dietary intake have long been suspected. Chou et al. (1993) have recently shown that caloric restriction alters the activities of several xenobiotic-metabolizing enzymes, including CYP1A1-dependent EROD and AHH activities and CYP1B1 dependent pentoxyresorufin-O-dealkylase (PROD) in DBA/2J or C57BL/6N mice. The nature of the interaction was not clear, since cytosolic AhR binding in both strains of mice was not increased and hepatic cytochrome CYP1A1 activity was increased in DBA/2J mice, a strain lacking normal AhR binding. The effects of caloric restriction, sex, and strain on CYP1A1 induction by TCDD are greater in females than in males of both strains, whereas the CYP isozymes induced in male DBA mice had less specificity toward 7-ethoxyresorufin than in C57BL/6N mice. TCDD induction was potentiated by caloric restriction in the DBA strain, indicating the interactive involvement of different regulatory mechanisms. In other studies, dietary fiber was shown to stimulate the fecal excretion of polychlorinated dibenzofurans (PCDF) and polychlorinated dibenzo-p-dioxins (PCDD) in rats (Morita et al., 1993). Chemical Interactions De Jongh et al. (1993) have presented evidence that toxicokinetic interactions occur in mixtures of PCDDs, PCDFs, and PCBs. In their studies, C57BL/6J mice were given single oral doses of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PnCDD), 1,2,3,6,7,8-hexachloro-dibenzo-p-dioxin (HxCDD), or 2,3,4,7,8-pentachlorodibenzofuran (PnCDF) (1.5-10.6 nmol/kg) as single compounds or in combination with 300 µmol/kg 2,2',4,4',5,5'-hexachlorobiphenyl (HxCB). Two other groups of mice received a mixture of the

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--> first three compounds, either with or without HxCB. The hepatic deposition and elimination of the compounds and their CYP1A1-dependent EROD activity were studied for 175 days. Interactive effects on the hepatic deposition of PnCDD were observed in most of the mixed-dose groups. For HxCDD and PnCDF, interactive effects were either very small or absent. No interactive effects were observed on the hepatic elimination rates of PnCDD, HxCDD, or PnCDF. Collectively, these data support the view that chemical interactions play critical roles in the disposition of chemical mixtures. In related studies, the hexachlorobiphenyl (HxCB) deposition in the livers of mothers and offspring was shown to be doubled in the presence of TCDD (DeJongh et al., 1994). HxCB co-administration did not influence hepatic TCDD deposition, suggesting that the mechanisms of interactions are different for both groups of compounds. Weber et al. (1992) have completed comparative toxicity studies in which male Sprague-Dawley rats were treated with an LD20, an LD50, and an LD80 of TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (penta-CDD), 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (hexa-CDD), 1,2,3,4,6,7,8,-heptachlorodibenzo-p-dioxin (hepta-CDD), respectively, and a mixture of the four homologues representing one-fourth of the previously established LD20, LD50, and LD80 for each hydrocarbon. While plasma tryptophan levels increased in a dose-dependent manner, EROD activity and liver weights in CDD-treated animals did not. These data suggest that a poor correlation exists between plasma tryptophan levels, a biomarker of acute toxicity, and EROD activity, a biomarker of AhR-mediated enzyme induction. It is clear that future studies must attempt to define the extent to which the presence of multiple chemicals in complex mixtures influences short-term versus long-term toxicity outcomes. Disease Outcomes and Mechanisms of Toxicity In this section, we summarize studies published during the period 1992-95 that investigated the toxic effects of TCDD. The mechanism of toxicity, if known, is also explained. Carcinogenicity Since relatively little data are available on carcinogenicity of TCDD in human populations with known TCDD body burdens, results from chronic feeding studies of laboratory animals continue to be used as the basis for human risk assessment. The results from animal bioassays were summarized in VAO. Recent investigations have focused on the mechanisms of carcinogenicity. These studies are summarized below. Mechanism of Toxicity The sex specificity of TCDD-induced hepatocellular carcinoma, along with the reduced tumor incidences of the pituitary, uterus, mammary gland, pancreas and adrenals in female relative to male rats, emphasize

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--> the view that hormonal control modulates TCDD carcinogenicity (Kociba et al., 1978). In addition to its ability to increase cancer rates in animals, TCDD can act as a promoter of tumor formation following initiation by other chemicals. To date, the consensus is that TCDD is not genotoxic and that its ability to influence the carcinogenic process is mediated via epigenetic events such as enzyme induction, cell proliferation, apoptosis, and intracellular communication. Studies investigating the epigenetic effects of TCDD are summarized below. Enzyme Induction The ability of TCDD to induce xenobiotic-metabolizing enzymes which can metabolize chemicals to reactive intermediates that are capable of causing injury has received considerable attention as a predictable biomarker of human exposures and/or carcinogenicity. The profiles of enzyme induction in experimental systems in vivo and in vitro often correlates with the patterns observed in human populations. Particular attention has been given to the induction of CYP1A1 and CYP1A2, and more recently to the newly characterized CYP1B1. The induction of CYP1A1 and CYP1A2, along with increases in phosphorylated forms of pp 32, 34, and 38, have been proposed as sensitive indicators of TCDD exposure (De Vito et al., 1994). In mice, the Ah gene battery also includes NAD(p)H:menadione oxidoreductase, aldehyde dehydrogenase, UDP-glucuronosyltransferase, and glutathione transferase, enzymes involved in phase II metabolism (Dunn et al., 1993; Takimoto et al., 1992; Vasiliou et al., 1992; Puga et al., 1992). The ability of TCDD to regulate the transcription of these genes is believed to occur via a receptor-mediated mechanism and is not restricted to the liver (Vasilou et al., 1993). The CYP1A1 gene is not inducible in certain cell types due to the presence of a putative repressor(s) that competes with the ligand/AhR complex for the responsive element or which otherwise precludes AhR/Arnt activity. Mechanistic studies with both wild-type and mutant cell lines have revealed a putative repressor of TCDD-dependent induction of CYP1A1 (Watson and Hankinson, 1992) that appears to be encoded by one of the Ah loci (Karenlampi et al., 1988). Basal levels of CYP1A1 transcription are controlled by a negative regulatory protein (Puga et al., 1992; Boucher et al., 1993; Sterling et al., 1993; Ou and Ramos, 1995), but this effect exhibits considerable species-specificity. For instance, three NRE binding proteins have been identified by electrophoretic mobility shift assays (EMSA) to participate in the negative regulation of the human CYP1A1 gene (Boucher et al., 1993). Normal human fibroblast nuclear extracts contain two constitutive protein-DNA complexes, which appear to be immunochemically distinct from the AhR (Gradin et al., 1993). Collectively, these data suggest that species- or cell-specific differences in responsiveness to TCDD and related chemicals involve interactions of multiple signals in the regulation of gene expression and raise important questions regarding heterogeneity of disease outcomes following herbicide exposures. Another complicating factor regarding the inducibility of CYP isozymes is

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--> recent data regarding the non-AhR inducibility of some of these enzymes. Daujat et al. (1992) showed that omeprazole, a benzimidazole derivative, induces both CYP1A1 and CYP1A2 in human liver via a nonreceptor mechanism. However, Quattrochi and Tukey (1993) have published a report that omeprazole does initiate AhR activation and that induction of the human CYP1A1 gene is indeed receptor-dependent. Cell Proliferation The effects of TCDD on hepatic epidermal growth factor receptor (EGFR) levels in a two-stage initiation/promotion model were recently investigated by Sewall et al. (1993). The doses employed encompassed the range used in bioassays to assess cancer potency for human health risk assessment. TCDD was administered bi-weekly by gavage to female Sprague-Dawley rats for 30 weeks following initiation by a single dose of diethylnitrosamine (DEN). Consistent with previous data, TCDD induced a decrease in EGFR in intact but not ovariectomized animals. A significant dose-dependent decrease in plasma membrane EGFR binding capacity was observed in initiated and noninitiated rats. The decrease in plasma membrane EGFR determined by equilibrium binding was confirmed by measurements of EGFR autophosphorylation and by immunohistochemical detection. Collectively, these results demonstrate that decreases of the EGFR by TCDD is ovarian-dependent and is a sensitive effect induced at dose levels associated with TCDD hepatocarcinogenicity in rodent bioassays. TCDD and HCDD had no effects on the proliferation of normal hepatocytes, but the labeling indices of enzyme-altered liver lesions were slightly enhanced by chemical treatment (Buchmann et al., 1994). Whether the selective, albeit moderate, increase in proliferation of enzyme-altered liver cells is sufficient to explain the promoting activity of dioxin, or whether additional factors such as effects of apoptosis are also important, remains to be established. Apoptosis Apoptosis (programmed cell death) has long been recognized as a normal process during organogenesis. More recently, apoptosis has been implicated as a key regulatory event in disorders of growth and differentiation, such as cancer. Selective apoptosis, in concert with cell-specific replication, may explain the unique promoting effects of different carcinogens such as TCDD, because an undesirable population of cells may be afforded a growth advantage (Marsman and Barrett, 1994). Studies to test this hypothesis will be useful in defining the role of apoptosis in TCDD-induced carcinogenesis. Intracellular Communication TCDD and two co-planar PCBs caused a rapid and sustained dose-dependent inhibition of intracellular communication in Hepa-1 cells (De Hann et al., 1994). The time course of inhibition of intracellular communication paralleled that of EROD induction, although the onset of communication inhibition preceded changes in EROD. A role for the AhR in the

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--> inhibition of intracellular communication was proposed based on the lack of inhibition in AhR-defective cells and the observation that α-naphthoflavone, an AhR antagonist, greatly reduced the TCDD effect. Immunotoxicity Extensive evidence has been published that halogenated aromatic hydrocarbons, including TCDD, polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polybrominated polyphenols (PBBs) exert toxic effects on the immune system (Kerkvliet and Burleson, 1994). The evidence is based on numerous studies of various animal species, including nonhuman primates, mice, rats, guinea pigs, rabbits, and chickens. A major target of halogenated aromatic hydrocarbons on the immune system appears to be the T-cell arm of the immune response. The finding that TCDD administration may result in thymic atrophy supports the concept that the immunotoxic effects are primarily mediated through the T cell (Kerkvliet and Burleson, 1994). Direct effects of TCDD on T cells in vitro, however, have not been demonstrated, suggesting that the action of TCDD and related chemicals may be indirect. A number of recent studies have focused on the interactions of halogenated aromatic hydrocarbons and cytokines. These soluble mediators are important in regulating the immune response. Based on its effect on the generation of T cells, it is reasonable to conclude that TCDD will increase susceptibility of experimental animals to challenge by pathogenic microorganisms that interact primarily with cell-mediated immunity. In support of this, it has been shown that TCDD exposure increases the susceptibility of mice and rats to challenge with intracellular pathogens such as Salmonella, Listeria , herpes virus, influenza virus, and Plasmodium. Similarly, growth of certain transplanted tumors is enhanced in mice treated with TCDD. On the other hand, T cells are usually required for antibody production. Increased susceptibility of mice to challenge by Streptococcus pneumoniae , an extracellular pathogen, may relate to impaired antibody production due to loss of T-cell help. In addition, investigations conducted by Neubert et al. (1994) indicate that TCDD under certain conditions appears to have no deleterious effect on the induced immune response. These investigators studied the proliferative capacity of marmoset lymphocytes during a secondary immune response following a tetanus vaccination. The animals were given an additional injection three months or one year following initial immunization. During this period, a proliferative response of lymphocytes to tetanus toxoid was documented. The investigators then studied the effect of TCDD on the booster response. Lymphocyte responses of four marmosets treated with TCDD were, in fact, greater than in control animals at the time of the second (one year) booster. Summarized below are recent animal studies, conducted in a variety of species, that support the finding that TCDD exposure can result in thymic atrophy and that this effect is primarily mediated through the T cell. In vitro evidence that this action of TCDD may be an indirect effect of TCDD on T cells is also summarized.

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--> Rice et al. (1995) treated female Fischer 344 rats with a single intraperitoneal dose of 0.3, 3.0, or 30 µg/kg TCDD or corn oil and examined cytotoxic T-cell activities 24 days after treatment. Syngeneic in vivo tumor-specific CTLs were generated that model cell-mediated immune reactions against neoplastically transformed self antigens. RT2, a virally-induced Fischer 344 rat glioma, and D74, an ethylnitrosurea-induced Fischer 344 rat glioma were used as targets. This immunological parameter was compared to body, thymic, and liver weights as well as liver ethoxyresorufin deethylase (EROD) activity on day 24 post-TCDD treatment. They found that Fischer 344 rats are very sensitive to TCDD. Immunotoxicity, indicated by severe thymic atrophy, was evident and dose-dependent. In contrast, there was no significant, consistent suppression of cytotoxic T-cell activity in a number of tumor targets, even at the highest dose tested (30 µg/kg dose). TCDD was reported to induce an increase in cytochrome P4501A1 (CYP1A1) activity at all doses tested. Earlier reports that low doses of TCDD (4 ng/kg/week) in the mouse suppressed the production of cytotoxic T cells were also not confirmed by Hanson and Smialowicz (1994). They did, however, find decreased thymic weight and cellularity at doses of 1.0 and 3.0 µg/kg/week. Similarly, suppression of antibody production was observed only at the higher doses. De Waal et al. (1993) exposed juvenile male Wistar rats to 150 µg/kg TCDD by oral intubation and killed them four or ten days later. TCDD was shown to retard the differentiation of the thymic epithelium. Specifically, a relative shift from immature to more mature cortical epithelial cells, as judged by electron microscopy, suggested an arrest in orderly cellular differentiation. In TCDD-induced thymic atrophy, the epithelial framework of the cortex becomes more compact, with focal aggregation of epithelial cells. These alterations in epithelial development and architecture may be responsible for defective intrathymic T-cell processing. Oughton et al. (1995) exposed female C57BL/6 mice to TCDD in a long-term study. Age- and TCDD-related changes in the phenotypes of splenic, thymic, and peripheral blood lymphocytes were investigated. When changes due to age were taken into account, TCDD treatment had no discernible effect on the total number of circulating T cells, B cells, and macrophages. There was, however, a small but statistically significant decrease in the frequency of the subpopulation of CD8 + cytotoxic T cells in the spleen following TCDD treatment. These changes were reflected by a significant decrease in the frequency of CD8+ T cells in the blood. Chronic exposure resulted in a small, statistically significant increase in the frequency of CD4CD8- thymic cells. These double negative cells probably represent immature T cells. A small but significant increase in T-cell receptor-bearing thymocytes was observed. TCDD also induced liver CYP1A1 microsomal enzymes. In conclusion, TCDD has been shown to suppress both cell-mediated immunity (CMI) and humoral immunity (HI) (Lundberg et al., 1992). TCDD prevents

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--> the maturation of the thymocytes to mature T cells by inducing differentation of thymic epithelial cells (Vanden Heuvel and Lucier, 1993). The suppression of CMI is most apparent in young animals (Faith, 1979). Suppression of HI occurs in older animals exposed to TCDD and results in an inhibition of B-lymphocyte differentiation into antibody-producing cells (Lundberg et al., 1992). Mechanism of Toxicity Studies indicate that the immunotoxic effects of TCDD and related substances are medicated through binding to the Ah receptor. This conclusion is based on reports that genetic variation at the Ah locus results in differing susceptibilities to TCDD immunotoxicity. Similarly, as demonstrated by Fernandez-Salguero et al. (1995), strains of mice lacking the Ah receptor are relatively resistant to TCDD-induced immunosuppression. By homologous recombination, the investigators constructed mice that were deficient in the Ah receptor. Almost half of the mice died shortly after birth. The survivors showed decreased accumulation of lymphocytes in the spleen and lymph nodes, but not in the thymus. The livers of the ''knock-out" Ah-receptor-deficient mice were reduced in size. These mice were unresponsive to TCDD induction of genes encoding P450 enzymes that catalyze the metabolism of foreign compounds. Thus, the Ah receptor appears to play an important role in mediating the immunotoxic effects of TCDD and in promoting the normal development of the immune system. The effect of TCDD on interleukin-mediated modulation of the immune response in (C57BL/6×C3H)F1 mice was studied by Karras, Conrad and Holsapple (1995). They hypothesized that the immunosuppression mediated by direct exposure of TCDD to B cells in vitro is due to an IL-4-like biological activity. Therefore, they studied the ability of TCDD to mimic the responses of B cells to IL-4, including upregulation of the major histocompatibility complex antigens of class II type, increases in the expression of the Fc receptor for IgE (CD23), and induction of immunoglobulin class switching. At concentrations that readily suppress B cell-proliferative and antibody-forming cell responses, TCDD failed to demonstrate any of the activities of IL-4. Furthermore, when TCDD was preincubated with B cells before addition of IL-4, no evidence of increased IL-4 activity was observed. In fact, TCDD preincubation resulted in decreased secretion of IgG1 and IgE from B cells stimulated to undergo immunoglobulin class switch by LPS and IL-4. Thus, it appears that TCDD inhibits the formation of fully differentiated B cells capable of secreting antibody and has no effects on class-switching events as such. It also appears that the observed immunosuppressive effects of TCDD on B-cell function cannot be explained by biological mimicry of the actions of IL-4. Takenaka et al. (1995) prepared cell cultures of peripheral blood monocytic cells and tonsil cells from healthy human donors between the ages of 21 and 48 years. They produced B-cell-rich populations by depleting T cells. The B cells were then stimulated by addition of IL-4 plus CD40, and the amount of IgE

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--> 6 Epidemiologic Studies In seeking evidence for associations between health outcomes and exposure to herbicides and TCDD, many different kinds of epidemiologic studies must be considered. Each study has varying degrees of strengths and weaknesses and contributes evidence to an association with the health outcomes considered in Chapters 7, 9, 10 and 11. There are three main groups of individuals studied with respect to herbicide exposure: those with occupational, environmental, and military exposures. The historical basis for the groups studied was examined in Chapter 2 of VAO. A description of how the articles to be reviewed were selected from the literature can be found in Appendix A of VAO. This chapter summarizes epidemiologic studies and reports reviewed by the committee. Included are studies published after VAO, studies that were not reviewed by the committee that wrote VAO, and studies that have been updated since to the publication of VAO. Tables 6-1, 6-2, and 6-3 present a brief overview of all of the epidemiologic studies reviewed for both VAO and this report. The summaries of the study present the study methods used, including, how the study subjects were ascertained; how the data were collected; the inclusion criteria; and how the exposure was determined, including 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid), chlorophenols, and the TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) contaminant. Additionally, the numbers in the study and comparison populations, when available, are given along with a brief description of the study in Tables 6-1, 6-2, and 6-3. No results are presented here; rather, the chapter provides a methodologic framework for the health outcome chapters that follow. Qualitative critique of the study design, population size, methods of data collection, case and control ascertainment, or

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--> quality of exposure assessment has been reserved for the individual health outcome chapters in which the results of these studies are discussed. The text and tables in this chapter are organized in three basic sections—occupational studies, environmental studies, and studies in Vietnam veterans—with subsections included under each heading. The studies focused on exposures to 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid), chlorophenols, and the TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) contaminant, 2,4-D, 2,4,5-T, 4-chloro-2-methylphenoxyacetic acid (MCPA), picloram, hexachlorophene, and chlorophenols, including trichlorophenol. In several instances, the investigators did not indicate which specific chemicals study participants were exposed to, or levels of exposure. Where available, details are given with regard to exposure assessment and how exposure was subsequently used in the analysis. The occupational section includes studies of production workers, agricultural/forestry workers (including herbicide/pesticide applicators), and paper/pulp workers, as well as case-control studies of specific cancers and the association with exposures to herbicides and related compounds in many of these occupations. The environmental section includes studies of populations exposed to excessive herbicides as a result of where they live, such as the residents of Seveso, Italy, Times Beach, Missouri, and the southern portion of Vietnam. The section on Vietnam veterans includes studies conducted in the United States by the Air Force, the Centers for Disease Control and Prevention (CDC), Department of Veterans Affairs (DVA, formerly the Veterans Administration), the American Legion, and the state of Michigan, as well as other groups. Studies of Australian veterans of Vietnam are also presented here. Occupational Studies Several occupational groups in the United States and elsewhere have been exposed to the types of herbicides used in Vietnam and, more specifically, to TCDD, a contaminant of some herbicides and other products. Occupational groups exposed to these chemicals include farmers, agricultural/forestry workers, herbicide sprayers, workers in chemical production plants, and workers involved in paper/pulp manufacturing. In addition, studies that use job titles as broad surrogates of exposure and studies that rely on disease registry data have been conducted. Exposure measures vary widely in these studies in terms of measurement, quantification, level of detail, confounding by other exposures, and individual versus surrogate or group (ecological) measures.

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--> TABLE 6-1 Epidemiologic Studies—Occupational Exposure Reference Study Design Description Study Group (N) Comparison Group (N)a Production Workers NIOSH New studies Egeland et al., 1994 Cohort Study of total serum testosterone and gondadotropin levels in chemical production workers exposed to dioxin, in same group as Calvert et al. (1991) 248 231 Calvert et al., 1994 Cross-sectional Study of porphyria cutanea tarda in same group as Calvert et al., (1991) 281 260 Studies reviewed in VAO Fingerhut et al., 1991 Cohort Cancer mortality in male workers from 12 plants producing TCDD-contaminated chemicals (1942-1984) compared to U.S. population 5,172 — Calvert et al., 1991 Cohort Study of workers employed at one of two plants manufacturing substances contaminated with TCDD 15 years or more prior to assessment of chronic bronchitis, COPD, ventilatory function, and thorax and lung abnormalities compared to neighborhood controls without exposure to TCDD 281 260 Calvert et al., 1992 Cohort Assessment of liver and gastrointestinal systems in same group as Calvert et al. (1991) 281 260 Alderfer et al., 1992 Cohort Assessment of psychological variables to determine depression in same group as Calvert et al. (1991) 281 260 Sweeney et al., 1993 Cohort Peripheral neuropathy in same group as Calvert et al. (1991) 281 260 Monsanto Zack and Suskind, 1980 Cohort Evaluation of mortality experience among employees with chloracne exposed to TCP process accident in 1949 at Monsanto compared to U.S. male population standard 121 — Zack and Gaffey, 1983 Cohort Study of mortality experience of all white male workers (1955-1977) employed at a Monsanto plant through December 31, 1977, compared to mortality of standardized U.S. population rates 884 — Suskind and Hertzberg, 1984 Cohort Evaluation of health outcomes (1979) at clinical examination among workers exposed to 2,4,5-T (1948-1969) compared to nonexposed workers at same Monsanto plant 204 163

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--> Moses et al., 1984 Cohort Study of health outcomes in Monsanto workers (1948-1969) with chloracne reported as a surrogate to 2,4,5-T exposure compared to health outcomes in workers without chloracne as surrogate for no exposure 117 109 Collins et al., 1993 Cohort Mortality of workers (through 1987) exposed and unexposed to dioxin between March 8, 1949, and November 22, 1949, as indicated by presence of chloracne, compared to local population mortality rates 122 With chloracne 632 Without chloracne — Dow New studies Bloeman et al., 1993 Cohort Additional years of follow-up of Bond et al. (1988) study cohort through 1986 878 (1) U.S. population; (2) 36,804 Unexposed workers Studies reviewed in VAO Ott et al., 1980 Cohort Mortality experience among workers exposed to 2,4,5-T in manufacturing (1950-1971) compared to mortality experience of U.S. white men 204 — Cook et al., 1980 Cohort Mortality experience (through 1978) of male workers involved in a chloracne incident (1964) from TCDD exposure compared to mortality experience of U.S. white men 61 — Bond, 1987 Cohort Extension of Cook et al. (1980) study, mortality through 1982 322 (1) U.S. white male population (2) 2,026 Employees without chloracne Bond et al., 1983 Cross-sectional Study of differences in workers potentially exposed and unexposed to TCDD during chemical production for (1) morbidity and (2) medical examination frequency between 1976 and 1978 (1) 183 (2) 114 (1) 732 (2) 456 Cook et al., 1986 Cohort Mortality experience (1940-1979) of men manufacturing chlorinated phenols compared to U.S. white men 2,189 — Ott et al., 1987 Cook et al., 1987 Cohort Expanded Cook et al. (1986) study an additional three years, through 1982 2,187 —

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--> Reference Study Design Description Study Group (N) Comparison Group (N)a Bond et al., 1989b Cohort Extension of Ott et al. (1987) study through 1984 2,187 — Bond et al., 1989a Cohort Study of incidence of chloracne among a cohort of workers potentially exposed to TCDD, and association with other risk factors 2,072 Internal comparison Bond et al., 1988 Cohort Study of mortality (through 1982) among workers potentially exposed to 2,4-D (1945-1983) compared to U.S. white males and all other male employees not exposed 878 (1) U.S. white male population (2) 36,804 Employees not exposed Sobel et al., 1987 Case-control Study of STS among Dow Chemical employees (1940-1979) compared to employees without STS for possible association with several chemical exposures 14 126 Townsend et al., 1982 Cohort Study of adverse reproductive outcomes among wives of Dow Chemical employees potentially exposed to TCDD (1939-1975) compared to reproductive outcomes among wives whose husbands were not exposed 370 345 Other Chemical Plants New studies Zober et al., 1994 Cohort Morbidity experience in the same group as Zober et al. (1990) 158 161 Lynge et al., 1993 Cohort Cancer incidence in the same group as Lynge (1985), with follow-up extended through 1987 3,390 Men 1,071 Women — Kogevinas et al., 1993 Cohort Cancer incidence and mortality experience of female workers in seven countries potentially exposed to chlorophenoxy herbicides, chlorophenols, and dioxin compared to national death rates and cancer incidence rates 701 — Kogevinas et al., 1992 Cohort Study of mortality from STS and malignant lymphomas in an international cohort of production workers and herbicide sprayers (same group as Saracci et al., 1991) 14,439 (13,482 exposed, 416 probably exposed, 541 with unknown exposure) 3,951 Non-exposed employees Kogevinas et al., 1995 Case-control Two nested case-control studies of the relationship between STS and NHL and occupational exposures in members of the IARC cohort STS: 11 cases NHL: 32 cases 5 Controls per case

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--> Studies reviewed in VAO Thiess et al., 1982 Cohort Study of mortality experience among BASF employees potentially exposed to TCDD during November 17, 1953, accident compared to population and other workers not exposed 74 External controls 180,000 town 1.8 million district 60.5 million Federal Republic of Germany Two groups of 74 each from other cohort studies Zober et al., 1990 Cohort Mortality experience of workers exposed to TCDD (1954-1987) at BASF plant compared to population of Federal Republic of Germany 247 — Manz et al., 1991 Cohort Mortality experience of workers (1952-1984) at Hamburg plant of Boehringer exposed to TCDD compared to national mortality and workers from another company 1,184 Men (a) Population (b) 3,120 Gas workers Saracci et al., 1991 Cohort Study of mortality experience of 20 international cohorts of herbicide sprayers and production workers compared to mortality experience expected for the nation 399 Women 16,863 Men 1,527 Women — — Coggon et al., 1986 Cohort Study of mortality experience (through 1983) among workers manufacturing and spraying MCPA (1947-1975) compared to expected numbers of deaths among men in England and Wales and for rural areas 5,754 — Coggon et al., 1991 Cohort Mortality experience among four cohorts of workers potentially exposed (1963-1985) to phenoxy herbicides and chlorophenols compared to national (England and Wales) expected numbers and to the local population where factory is located 1,104 Factory A 271 Factory B 345 Factory C 519 Factory D — Jennings et al., 1988 Cohort Assessment of immunological abnormalities among workers exposed to TCDD during accident manufacturing 2,4,5-T compared to matched controls 18 15 May, 1982, 1983 Cohort Health outcomes among workers exposed and probably exposed to TCDD following a 1968 accident compared to unexposed workers 41 Exposed 54 Possibly exposed 31

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--> Reference Study Design Description Study Group (N) Comparison Group (N)a Bueno de Mesquita et al., 1993 Cohort Mortality experience of production workers exposed to phenoxy herbicides and chlorophenols in the Netherlands compared to national rates 2,310 — Bashirov, 1969 Cross-sectional Descriptive results of examination of workers involved in production of herbicides and study of workers at examination of cardiovascular and digestive systems compared to unexposed controls 292 (descriptive) 50 (examined) 20 (examined) Lynge, 1985 Cohort Study of cancer incidence among Danish workers exposed to phenoxyherbicides compared to expected results from the general population 3,390 Men 1,069 Women — Pazderova-Vejlupkova et al., 1981 Descriptive Study of development of TCDD intoxication among men in Prague (1965-1968) 55 No comparison group Poland et al., 1971 Cross-sectional Assessment of PCT, chloracne, hepatotoxicity, and neuropsychiatric symptoms among 2,4-D and 2,4,5-T workers compared to other plant workers 73 Total 20 Administrators 11 Production supervisors 28 Production workers 14 Maintenance workers Internal comparison Thomas, 1987 Cohort Assessment of mortality experience as of January 1, 1981, for white men employed in fragrance and flavors plant with possible exposure to TCDD compared to U.S. white men, and for cancers compared to local men 1,412 — Agricultural/Forestry Workers 1. COHORT STUDIES Agricultural Workers New studies Blair et al., 1993 Cohort Study of causes of death, including cancer, among farmers in 23 states (1984-1988) 119,648 White men 2,400 White women 11,446 Nonwhite men 2,066 Nonwhite women —

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--> Dean, 1994 Cohort Study of mortality from brain and hematopoietic cancers of agricultural workers compared to nonagricultural workers in Ireland (1971-1987) (population size unclear) — Morrison et al., 1994 Cohort Update of mortality experience in Wigle et al. (1990) cohort, through 1987, with addition of farmers from Alberta and Manitoba. 155,547 — Semenciw et al., 1993 Cohort Study of multiple myeloma mortality of male farmers compared to male population of the three prairie provinces of Canada (1971-1987) 155,547 — Semenciw et al., 1994 Cohort Study of leukemia mortality in same group as Morrison et al. (1993) 155,547 — Senthilselvan et al., 1992 Cross-sectional Study of the association between pesticide exposure and asthma in male farmers 1,939 No comparison group Studies reviewed in VAO Burmeister, 1981 Cohort Study of mortality of farmers compared to nonfarmers in Iowa (1971-1978) 6,402 13,809 Wigle et al., 1990 Cohort Mortality experience from NHL of male farmers 35 years or older (1971-1985) in Saskatchewan, Canada, compared to age- and period-specific mortality rates expected for Saskatchewan males 69,513 — Morrison et al., 1993 Cohort Mortality experience of male Canadian farmers 45 years or older in Manitoba, Saskatchewan, and Alberta, Canada (1971-1987), compared to Canadian prairie province mortality rates 145,383 — Morrison et al., 1992 Cohort Mortality experience of male farmers 35 years or older (1971-1987) compared to Canadian prairie province rates 155,547 — Ronco et al., 1992 Cohort Study of cancer incidence (1970-1980) among male and female Danish farm workers 15-74 years old compared to expected numbers of cancers among persons economically active, and study of cancer mortality (November 1981-April 1982) among male and female Italian farmers 18-74 years old compared to persons in other occupational groups No Ns given No Ns given Corrao et al., 1989 Cohort Study of cancer incidence among male farmers licensed (1970-1974) to use pesticides compared to number of cancers expected among licensed nonusers 642 18,839 Lerda and Rizzi, 1991 Cohort Study of farmers exposed to 2,4-D as measured in urine compared to men unexposed for differences in sperm volume, death, count, motility, and abnormalities between March and June 1989 32 25

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--> Reference Study Design Description Study Group (N) Comparison Group (N)a Hansen et al., 1992 Cohort Study of cancer incidence among male and female Danish gardeners compared to incidence expected among the general population 4,015 859 Women 3,156 Men — Wiklund, 1983 Cohort Study of cancer incidence (diagnosed 1961-1973) among agricultural workers in Sweden compared to rates expected from the 1960 population census 19,490 — Wiklund and Holm, 1986 Cohort STS incidence among agricultural and forestry workers in Sweden compared to the general population of men, 1960 census 354,620 1,725,845 Wiklund et al., 1998a Cohort Malignant lymphoma incidence among agricultural and forestry workers in Sweden compared to the general population of men, 1960 census 354,620 1,725,845 Eriksson et al., 1992 Cohort Study of incidence of NHL, HD, and multiple myeloma (1971-1984) among selected occupational groups in Swedish men and women compared to expected rates of disease in general population Number in occupational group unknown — Forestry Workers Green, 1987 Cohort Suicide experience in a cohort of Canadian forestry workers by number of years in forestry trade as a surrogate for exposure to phenoxy herbicides compared to population 1,222 — Green, 1991 Cohort Mortality experience of male forestry workers (1950-1982) in Ontario compared to the expected mortality of the male Ontario population 1,222 — van Houdt et al., 1983 Cross-sectional Study of acne and liver dysfunction in a select group of Dutch forestry workers exposed to 2,4,5-T and unexposed 54 54 Herbicide/Pesticide Sprayers New studies Asp et al., 1994 Cohort Mortality and cancer morbidity experience of male chlorophenoxy herbicide applicators (same cohort as Riihimaki et al., 1982 and 1983) in Finland (1955-1971), through 1989, compared to general population rates for morbidity and mortality. 1,909 — Garry et al., 1994 Cross-sectional Evaluation of health outcomes resulting from exposure to pesticides by male pesticide appliers in Minnesota 719 No comparison group

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--> Studies reviewed in VAO Axelson and Sundell, 1974 Cohort Study of mortality and cancer incidence among cohorts of Swedish railroad workers spraying herbicides (>45 days) compared to the expected number of deaths (1957-1972) from Swedish age- and sex-specific rates 348 Total herbicide exposure 207 Phenoxy acids and combinations 152 Amitrole and combinations 28 Other herbicides and combinations — Axelson et al., 1980 Cohort Additional years of follow-up to cohort established in Axelson and Sundell (1974) 348 — Blair, 1983 Cohort Mortality experience of white male Florida pesticide applicators compared to U.S. and Florida men 3,827 — Riihimaki et al., 1982 Cohort Study of mortality among herbicide applicators exposed to 2,4-D and 2,4,5-T in Finland compared to mortality expected in the population 1,926 — Riihimaki et al., 1983 Cohort Cancer morbidity and mortality in cohort (Riihimaki et al., 1982) through 1980 1,926 — Smith et al., 1981 Cohort Study of chemical applicators (1973-1979) in New Zealand compared to agricultural contractors for differences in adverse reproductive outcomes 459 422 Smith et al., 1982 Cohort Study of adverse reproductive outcomes among chemical applicators and agricultural contractors by category of exposure: none; chemicals not 2,4,5-T; and 2,4,5-T 113 Pregnancies (chemicals not 2,4,5-T) 486 Pregnancies (2,4,5-T) 401 Pregnancies (not exposed) Wiklund, 1987 Cohort Risk of HD and NHL among Swedish pesticide applicators from date of license through 1982 compared to expected number of cases in the total population 20,245 — Wiklund et al., 1988b Cohort Risk of STS in Wiklund et al. (1987) cohort through 1984 20,245 — Wiklund et al., 1989a Cohort Risk of cancer in Wiklund et al. (1987) cohort through 1982 20,245 — Wiklund et al., 1989b Cohort Risk of STS, HD, and NHL in Wiklund et al. (1987) cohort through 1984 20,245 — Swaen et al., 1992 Cohort Cancer mortality experience (through 1987) among Dutch male herbicide applicators licensed before 1980 compared to the total male Dutch population 1,341 — Bender et al., 1989 Cohort Cancer mortality of Minnesota highway maintenance workers compared to expected numbers based on white Minnesota men 4,849 —

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--> Reference Study Design Description Study Group (N) Comparison Group (N)a Barthel, 1981 Cohort Study of male agricultural production workers (1948-1972) for incidence of cancer compared to incidence rates expected in the population 1,658 — 2. CASE-CONTROL STUDIES New studies Persson et al., 1993 Case-control Study of risk factors potentially associated with HD and NHL in males identified from the Regional Cancer Registry in Sweden HD: 31 NHL: 93 204 Hardell et al., 1994 Case-control Study of the association between occupational exposures and parameters related to NHL in white males in Sweden 105 335 Brown et al., 1993 Case-control Population-based case-control study of multiple myeloma in Iowa men for association with pesticide exposures 173 650 Zahm et al., 1993 Case-control Study of NHL and exposure to pesticides in white women diagnosed with NHL between July 1, 1983, and June 30, 1986 206 824 Mellemgaard et al., 1994 Case-control Study of cases of renal-cell carcinoma (20-79 years) in Denmark compared to population-based sample without cancer for identification of occupational risk factors 365 396 McDuffie et al., 1990 Case-control Study of pesticide exposure in male cases of primary lung cancer in Saskatchewan compared to control subjects matched by age, sex, and location of residence 273 187 Nurminen et al., 1994 Case-control Study of infants with structural defects born to mothers engaged in agricultural work during the first trimester of pregnancy compared to infants with structural defects born to mothers who did not engage in agricultural work during the first trimester 1,306 1,306 Semchuk, 1993 Case-control Study of cases of Parkinson's disease (36-90 years) in Canada compared to population-based sample for association with occupational exposure to herbicides and other exposures 75 Men 55 Women 150 Men 110 Women Studies reviewed in VAO Hardell and Sandstrom, 1979 Case-control Study of male cases of STS (26-80 years) diagnosed between 1970 and 1977 in northern Sweden compared to population- based sample without cancer for association with occupational exposure to phenoxyacetic acids and chlorophenols 52 206 Eriksson et al., 1979, 1981 Case-control Study of cases of STS diagnosed between 1974 and 1978 in southern Sweden compared to population-based sample without cancer for association with occupational exposure to phenoxyacetic acids and chlorophenols 110 219