Using residential histories in case-control analysis of lung cancer and mountaintop removal coal mining in Central Appalachia.

Population-based ecological and cross-sectional studies have observed high risk for several cancers in areas of Central Appalachia where mountaintop removal coal mines operate. Case-control studies could provide stronger evidence of such relationships, but misclassification of exposure is likely when based on current residence, since individuals could have inhabited several residences with varying environmental exposures over many years. To address this, we used residential histories for individuals enrolled in a previous case-control study of lung cancer to assess residential proximity to mountaintop removal coal mining over a 30-year period, using both survey data and proprietary data from LexisNexis, Inc. Supplementing the survey data with LexisNexis data improved precision and completeness of geographic coordinates. Final logistic regression models revealed higher odds of high exposure among cases. These findings suggest that living in close proximity to mountaintop removal coal mining sites could increase risk for lung cancer, after adjusting for other relevant factors.

Expression of stimulator of Fe transport is not enhanced in Hfe knockout mice.

Hfe knockout (-/-) mice recapitulate many of the biochemical abnormalities of hereditary hemochromatosis (HH), but the molecular mechanisms involved in the etiology of iron overload in HH remain poorly understood. It was found previously that livers of patients with HH contained 5-fold higher SFT (stimulator of Fe transport) mRNA levels relative to subjects without HH. Because this observation suggests a possible role for SFT in HH, we investigated SFT mRNA expression in Hfe(-/-) mice. The 4- and 10-wk-old Hfe(-/-) mice do not have elevated levels of hepatic SFT transcripts relative to age-matched Hfe(+/+) mice, despite having 2.2- and 3.3-fold greater hepatic nonheme iron concentrations, respectively. Northern blot analyses of various mouse tissues revealed that SFT is widely expressed. The novel observation that SFT transcripts are abundant in brain prompted a comparison of SFT transcript levels and nonheme iron levels in the brains of Hfe(+/+) and Hfe(-/-) mice. Neither SFT mRNA levels nor nonheme iron levels differed between groups. Further comparisons of Hfe(-/-) and Hfe(+/+) mouse tissues revealed no significant differences in SFT mRNA levels in duodenum, the site of increased iron absorption in HH. Important distinctions between Hfe(-/-) mice and HH patients include not only differences in the relative rate and magnitude of iron loading but also the lack of fibrosis and phlebotomy treatment in the knockout animals.

Uroporphyria in Hfe mutant mice given 5-aminolevulinate: a new model of Fe-mediated porphyria cutanea tarda.

Porphyria cutanea tarda (PCT), a liver disease with skin lesions caused by excess liver production of uroporphyrin (URO), is associated with consumption of alcoholic beverages or estrogens, and moderate iron overload. Recently, it has been shown that many PCT patients carry mutations in the HFE gene, which is responsible for hereditary hemochromatosis. Mice homozygous for either the null mutation in the Hfe gene or the C282Y missense mutation rapidly accumulate hepatic parenchymal iron similar to patients with hemochromatosis. Here we investigated whether disruption of the murine Hfe gene would result in hepatic uroporphyria. Mice homozygous for the Hfe-null mutation accumulated high levels of hepatic URO when fed 5-aminolevulinate (ALA). Hfe (+/-) mice also accumulated hepatic URO when fed ALA, but at a much slower rate. The amount of accumulated URO in the null mutant mice was similar to that in wild-type mice treated with iron carbonyl in the diet, or injected with iron dextran. Iron in both wild-type and Hfe (+/-) mice was mostly in Kupffer cells. In contrast, Hfe (-/-) mice had considerable parenchymal iron deposition as well, in a pattern similar to that observed in wild-type mice treated with iron carbonyl. URO accumulation was accompanied by 84% and 33% decreases in hepatic uroporphyrinogen decarboxylase activities in Hfe (-/-) and Hfe (+/-) mice, respectively. No increases in CYP1A2 or other cytochrome P450s were detected in the Hfe-null mutant mice. We conclude that this experimental model of uroporphyria is a valid model for further investigations into the mechanism of PCT.

Expression of the DMT1 (NRAMP2/DCT1) iron transporter in mice with genetic iron overload disorders.

Iron overload is highly prevalent, but its molecular pathogenesis is poorly understood. Recently, DMT1 was shown to be a major apical iron transporter in absorptive cells of the duodenum. In vivo, it is the only transporter known to be important for the uptake of dietary non-heme iron from the gut lumen. The expression and subcellular localization of DMT1 protein in 3 mouse models of iron overload were examined: hypotransferrinemic (Trf(hpx)) mice, Hfe knockout mice, and B2m knockout mice. Interestingly, in Trf(hpx) homozygotes, DMT1 expression was strongly induced in the villus brush border when compared to control animals. This suggests that DMT1 expression is increased in response to iron deficiency in the erythron, even in the setting of systemic iron overload. In contrast, no increase was seen in DMT1 expression in animals with iron overload resembling human hemochromatosis. Therefore, it does not appear that changes in DMT1 levels are primarily responsible for iron loading in hemochromatosis.

A mouse model of familial porphyria cutanea tarda.

Approximately one-third of patients with porphyria cutanea tarda (PCT), the most common porphyria in humans, inherit a single mutant allele of the uroporphyrinogen decarboxylase (URO-D) gene. PCT associated with URO-D mutations is designated familial PCT. The phenotype is characterized by a photosensitive dermatosis with hepatic accumulation and urinary excretion of uroporphyrin and hepta-carboxylic porphyrins. Most heterozygotes for URO-D mutations do not express a porphyric phenotype unless hepatic siderosis is present. Hemochromatosis gene (HFE) mutations are frequently found when the phenotype is expressed. We used homologous recombination to disrupt one allele of murine URO-D. URO-D(+/-) mice had half-wild type (wt) URO-D protein and enzymatic activity in all tissues but did not accumulate hepatic porphyrins, indicating that half-normal URO-D activity is not rate limiting. When URO-D(+/-) mice were injected with iron-dextran and given drinking water containing delta-aminolevulinic acid for 21 days, hepatic porphyrins accumulated, and hepatic URO-D activity was reduced to 20% of wt. We bred mice homozygous for an HFE gene disruption (HFE(-/-)) to URO-D(+/-) mice, generating mice with the URO-D(+/-)/HFE(-/-) genotype. These animals developed a porphyric phenotype by 14 weeks of age without ALA supplementation, and URO-D activity was reduced to 14% of wt. These data indicate that iron overload alone is sufficient to reduce URO-D activity to rate-limiting levels in URO-D(+/-) mice. The URO-D(+/-) mouse serves as an excellent model of familial PCT and affords the opportunity to define the mechanism by which iron influences URO-D activity.

The Nramp2/DMT1 iron transporter is induced in the duodenum of microcytic anemia mk mice but is not properly targeted to the intestinal brush border.

Microcytic anemia (mk) mice and Belgrade (b) rats are severely iron deficient because of impaired intestinal iron absorption and defective iron metabolism in peripheral tissues. Both animals carry a glycine to arginine substitution at position 185 in the iron transporter known as Nramp2/DMT1 (divalent metal transporter 1). DMT1 messenger RNA (mRNA) and protein expression has been examined in the gastrointestinal tract of mk mice. Northern blot analysis indicates that, by comparison to mk/+ heterozygotes, mk/mk homozygotes show a dramatic increase in the level of DMT1 mRNA in the duodenum. This increase in RNA expression is paralleled by a concomitant increase of the 100-kd DMT1 isoform I protein expression in the duodenum. Immunohistochemical analyses show that, as for normal mice on a low-iron diet, DMT1 expression in enterocytes of mk/mk mice is restricted to the duodenum. However, and in contrast to normal enterocytes, little if any expression of DMT1 is seen at the apical membrane in mk/mk mice. These results suggest that the G185R mutation, which was shown to impair the transport properties of DMT1, also affects the membrane targeting of the protein in mk/mk enterocytes. This loss of function of DMT1 is paralleled by a dramatic increase in expression of the defective protein in mk/mk mice. This is consistent with a feedback regulation of DMT1 expression by iron stores. (Blood. 2000;96:3964-3970)

Genes that modify the hemochromatosis phenotype in mice.

Hereditary hemochromatosis (HH) is a prevalent human disease caused by a mutation in HFE, which encodes an atypical HLA class I protein involved in regulation of intestinal iron absorption. To gain insight into the pathogenesis of hemochromatosis, we have bred Hfe knockout mice to strains carrying other mutations that impair normal iron metabolism. Compound mutant mice lacking both Hfe and its interacting protein, beta-2 microglobulin (B2m), deposit more tissue iron than mice lacking Hfe only, suggesting that another B2m-interacting protein may be involved in iron regulation. Hfe knockout mice carrying mutations in the iron transporter DMT1 fail to load iron, indicating that hemochromatosis involves iron flux through DMT1. Similarly, compound mutants deficient in both Hfe and hephaestin (Heph) show less iron loading than do Hfe knockout mice, indicating that iron absorption in hemochromatosis involves the function of Heph as well. Finally, compound mutants lacking Hfe and the transferrin receptor accumulate more tissue iron than do mice lacking Hfe alone, consistent with the idea that interaction between these two proteins contributes to the control of normal iron absorption. In addition to providing insight into the pathogenesis of HH, our results suggest that each of these genes might be a candidate modifier of the human hemochromatosis phenotype.

Risk factors for conduct disorder among Navajo Indian men and women.

OBJECTIVES: To describe the risk factors for conduct disorder before age 15 among Navajo Indians. METHODS: The study was based on a survey of a stratified random sample of adult Navajo Indians between the ages of 21 and 65 living on and adjacent to two different areas of the Navajo Reservation. There were 531 male and 203 female respondents. The average age (SD) of the men was 38.7 (10.5) years and of the women 35.5 (9.0) years. Conduct disorder was diagnosed retrospectively using the Diagnostic Interview Schedule first developed for the Epidemiological Catchment Area study. The responses were combined into a continuous scale. RESULTS: Significant risk factors for increased scores on the conduct disorder scale were: histories of physical and sexual abuse in childhood; abusive maternal drinking; a small number of households per camp; younger age; and being male rather than female. Measures of social status and religion in which subjects were raised were not significant. CONCLUSIONS: Many of the risk factors that are associated with conduct disorder in other populations are also risk factors in the Navajo population. There is suggestive evidence that some of these risk factors have become more common since World War II, raising the possibility that conduct disorder has become more prevalent, as is thought to be the case nationwide.

The C282Y mutation causing hereditary hemochromatosis does not produce a null allele.

Targeted mutagenesis was used to produce two mutations in the murine hemochromatosis gene (Hfe) locus. The first mutation deletes a large portion of the coding sequence, generating a null allele. The second mutation introduces a missense mutation (C282Y) into the Hfe locus, but otherwise leaves the gene intact. This mutation is identical to the disease-causing mutation in patients with hereditary hemochromatosis. Mice carrying each of the two mutations were bred and analyzed. Homozygosity for either mutation results in postnatal iron loading. The effects of the null mutation are more severe than the effects of the C282Y mutation. Mice heterozygous for either mutation accumulate more iron than normal controls. Interestingly, although liver iron stores are greatly increased, splenic iron is decreased. We conclude that the C282Y mutation does not result in a null allele.

Transferrin receptor is necessary for development of erythrocytes and the nervous system.

Plasma iron circulates bound to transferrin (Trf), which solubilizes the ferric ion and attenuates its reactivity. Diferric Trf interacts with cell-surface Trf receptor (Trfr) to undergo receptor-mediated endocytosis into specialized endosomes. Endosomal acidification leads to iron release, and iron is transported out of the endosome through the activity of divalent metal transporter 1 (DMT1, formerly Nramp2), a transmembrane iron transporter that functions only at low pH. Trf and Trfr then return to the cell surface for reuse, completing a highly efficient cycle. Although the Trf cycle is assumed to be the general mechanism for cellular iron uptake, this has not been validated experimentally. Mice with hypotransferrinaemia (hpx) have little or no plasma Trf. They have severe anaemia, indicating that the Trf cycle is essential for iron uptake by erythroid cells. Other hpx tissues, however, are generally normal, and there is a paradoxical increase in intestinal iron absorption and iron storage. To test the hypothesis that the Trf cycle has unique importance for erythropoiesis, we disrupted the Trfr gene in mice. This results in elimination of the Trf cycle, but leaves other Trf functions intact. Mice lacking Trfr have a more severe phenotype than hpx mice, affecting both erythropoiesis and neurologic development. Furthermore, haploinsufficiency for Trfr results in impaired erythroid development and abnormal iron homeostasis.

Molecular insights into mechanisms of iron transport.

The past 3 years have witnessed extraordinary progress in our understanding of mammalian iron transport and homeostasis. The first transmembrane iron transporter has been found. Mutations in this protein, in two animal models with iron-transport defects, have helped to define the roles of this protein in vivo. The gene defective in patients with hereditary hemochromatosis has been identified, and much has been learned about the structure and function of its gene product. Finally, our ability to make a molecular diagnosis of hereditary hemochromatosis has called attention to new iron-loading disorders, including African iron overload and juvenile hemochromatosis.

Alcohol dependence and conduct disorder among Navajo Indians.

OBJECTIVE: The purpose of this study is to examine the association between conduct disorder before age 15 and subsequent alcohol dependence, and to describe the lifetime prevalence of alcohol dependence among Navajo Indian women and men. METHOD: This was a case-control design which included both men (n = 735) and women (n = 351) and in which the Diagnostic Interview Schedule was used for the diagnosis of the lifetime history of alcohol dependence and conduct disorder. Alcohol dependent cases were selected from inpatient and outpatient treatment programs (204 men, 148 women). Whenever possible, controls were matched for age, sex and community of residence and were randomly selected and interviewed until a nonalcohol dependent individual was found. Among the men, there were 374 alcohol dependent controls and 157 nonalcohol dependent controls. Among the women, the figures were 60 and 143, respectively. When combined, the controls comprise samples of the adult male and female populations from which estimates of lifetime prevalence of alcohol dependence, and of the amount of alcohol dependence in the population attributable to conduct disorder, may be inferred. RESULTS: Conduct disorder is a risk factor for alcohol dependence among both men and women. Lifetime prevalence of alcohol dependence in this population is high (70.4% for men and 29.6% for women), but the amount of alcohol dependence in the population attributable to conduct disorder is low. On the other hand, among the alcohol dependent, those with conduct disorder had the most severe alcohol- and nonalcohol-related problems. CONCLUSIONS: The potential limitations of the study are those common to case-control designs, especially biased recall by cases. There are also potential sampling biases among the controls. It is shown that none of the potential biases invalidate the findings, which support the hypothesis that in this population conduct disorder is a risk for alcohol dependence. The implications for primary prevention of alcohol dependence are discussed.

Boarding and public schools: Navajo educational attainment, conduct disorder, and alcohol dependency.

Many critics of United States government operated boarding schools for American Indians have asserted that the boarding school experience has lasting deleterious effects on personality development. Specifically, it has been suggested that a boarding school education is likely to lead to problems with alcohol in adulthood. To examine that assertion, data from interviews with over 1000 Navajos are analyzed concerning schooling, conduct disorder and the history of alcohol use. Consistent with data on the U.S. population generally, Navajo high school dropouts reported greater problems with alcohol than did graduates. Contrary to expectations, Navajos with a history of alcohol dependency were no more likely to have attended boarding schools than those who did not report patterns of alcohol dependency.

Alcohol dependence and domestic violence as sequelae of abuse and conduct disorder in childhood.

OBJECTIVES: To examine in the Navajo population: (1) the importance of childhood abuse as a risk factor for conduct disorder; (2) the importance of each form of abuse and conduct disorder as risk factors for alcohol dependence; and (3) the relative importance of each form of abuse, conduct disorder, and alcohol dependence as risk factors for being a perpetrator and/or victim of domestic violence. METHOD: The study is based on a case-control design. Cases (204 men and 148 women) between the ages of 21 and 65 were interviewed in alcohol treatment program and matched to community controls. There were two groups of controls: alcohol dependent (374 men, 60 women) and nonalcohol dependent (157 men, 143 women). When adjusted for stratification by age, community of residence, and sex, the combined control groups comprise a representative sample of the Navajo male and female population 21-65 years of age. RESULTS: The prevalence of physical and sexual abuse before age 15 is within limits observed in other populations. Each form of abuse is a risk factor for conduct disorder. Along with conduct disorder, physical abuse is a risk factor for alcohol dependence. Physical abuse and alcohol dependence are independent risk factors for being involved in domestic violence as both perpetrator and victim. There appears to have been no secular trend in the incidence of childhood abuse over the past several generations, but there is suggestive evidence that domestic violence has become more common. CONCLUSIONS: Physical abuse is a significant risk factor for alcohol dependence as well as for domestic violence independent of the effects of alcohol abuse. The effects of sexual abuse with regard to both domestic violence and alcohol dependence do not appear to be significant.

A prospective study of isolation and mortality in a cohort of elderly Navajo Indians.

The prospective association between social isolation and mortality in a sample of 271 elderly Navajo Indian men and women living on their reservation in northern Arizona is reported. The follow-up period averaged three years from the time of interview in 1982-3. Self-reported level of physical functioning was predictive of mortality. Of the psycho-social measures, only marital status among men was predictive of increased risk of death, with the unmarried being at higher risk than the married. These results are attributed to the fact that Navajo society is traditionally matrilineal and matrilocal, with the mother-daughter bond being especially significant and with men being relatively more peripheral than their wives to the kin group. Thus unmarried men are far more likely to be isolated from kin than unmarried women and married people of either sex.

A suicide prevention program for Hopi youth.

In recent years, Hopi Indians have been concerned about what they believe are rising suicide rates especially among teenagers and young adults. A review of 30 years of Hopi suicides reveals that: (a) although it is possible that rates are rising, it is more likely that they are relatively stable; (b) high age specific rates for those between 15 and 34 years of age is not a recent phenomenon; (c) the individuals at risk for suicide and for alcohol abuse are the children of parents who made traditionally disapproved marriages, i.e. intertribal, intermesa, and between clans of disparate social status. By labeling the parents as deviant the community creates 'primary' deviance in the second generation. To be successful, a suicide program must not be designed specifically for troubled adolescents. Nor can it identify the problem as caused by either acculturation or traditional culture. The proposed program and constraints placed upon its implementation are discussed.

The prevalence of hypertension among elderly Navajos: a test of the acculturative stress hypothesis.

This is a study of the prevalence of hypertension among a sample of Navajo Indians 65 years of age and above. It is not clear whether prevalence has increased over the past generation in this age group. When men and women are compared, conventional measures of "acculturation" are related to hypertension among women but not among men. The differences between men and women seem most probably related to differences in the situation of men and women within both Navajo and Anglo-American society. Several alternative explanations are discussed as well.