Biochar application to a contaminated soil reduces the availability and plant uptake of zinc, lead and cadmium.

Heavy metals in soil are naturally occurring but may be enhanced by anthropogenic activities such as mining. Bio-accumulation of heavy metals in the food chain, following their uptake to plants can increase the ecotoxicological risks associated with remediation of contaminated soils using plants. In the current experiment sugar cane straw-derived biochar (BC), produced at 700 °C, was applied to a heavy metal contaminated mine soil at 1.5%, 3.0% and 5.0% (w/w). Jack bean (Canavalia ensiformis) and Mucuna aterrima were grown in pots containing soil and biochar mixtures, and control pots without biochar. Pore water was sampled from each pot to confirm the effects of biochar on metal solubility, whilst soils were analyzed by DTPA extraction to confirm available metal concentrations. Leaves were sampled for SEM analysis to detect possible morphological and anatomical changes. The application of BC decreased the available concentrations of Cd, Pb and Zn in 56, 50 and 54% respectively, in the mine contaminated soil leading to a consistent reduction in the concentration of Zn in the pore water (1st collect: 99 to 39 µg L(-1), 2nd: 97 to 57 µg L(-1) and 3rd: 71 to 12 µg L(-1)). The application of BC reduced the uptake of Cd, Pb and Zn by plants with the jack bean translocating high proportions of metals (especially Cd) to shoots. Metals were also taken up by Mucuna aterrima but translocation to shoot was more limited than for jack bean. There were no differences in the internal structures of leaves observed by scanning electron microscopy. This study indicates that biochar application during mine soil remediation reduce plant concentrations of potential toxic metals.

Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats.

Gene therapy for cystic fibrosis (CF) will require the safe transfer of CFTR cDNA to airway epithelia in vivo. We showed previously that a recombinant adenovirus, Ad2/CFTR-1, expresses CFTR in vitro. As adenovirus rarely integrates, treatment will require repeated vector administration. We applied Ad2/CFTR-1 to intrapulmonary airway epithelia of cotton rats and nasal epithelia of Rhesus monkeys. In both species we detected CFTR mRNA and protein after repeated administration and in monkeys, protein was detected six weeks after repeat administration. The vector did not replicate and was rapidly cleared. Despite an antibody response, there was no evidence of a local or systemic inflammatory response after repeat administration. These data indicate that repetitive administration of Ad2/CFTR-1 is both safe and efficacious.

Activity and stability of bifunctional perovskite/carbon-based electrodes for the removal of antipyrine by electro-Fenton process.

Bifunctional perovskite/carbon-black(CB)/polytetrafluoroethylene(PTFE) electrodes for electro-generation and catalytic decomposition of hydrogen peroxide to oxidizing hydroxyl radicals have been fabricated. These electrodes were tested for electroFenton (EF) removal of antipyrine (ANT) as a model antipyretic and analgesic drug. The influence of the binder loading (20 and 40 wt % PTFE) and type of solvent (1,3-dipropanediol and water) was studied for the preparation of CB/PTFE electrodes. The electrode prepared with 20 wt % PTFE and water exhibited a low impedance and remarkable H2O2 electro-generation (about 1 g/L after 240 min, a production rate of ca. 6.5 mg/h·cm2). The incorporation of perovskite on CB/PTFE electrodes was also studied following two different methods: i) direct deposition on the CB/PTFE electrode surface and ii) addition in the own CB/PTFE/water paste used for the fabrication. Physicochemical and electrochemical characterization techniques were used for the electrode's characterization. The dispersion of perovskite particles in the own electrode matrix (method ii) exhibited a higher EF performance than the immobilisation onto the electrode surface (method i). EF experiments at 40 mA/cm2 and pH 7 (non-acidified conditions) showed ANT and TOC removals of 30% and 17%, respectively. The increase of current intensity up to 120 mA/cm2 achieved the complete removal of ANT and 92% of TOC mineralisation in 240 min. The bifunctional electrode also proved high stability and durability after 15 h of operation.

Trends of asthma mortality in Galicia from 1993 to 2007.

INTRODUCTION: In the past few years, deaths due to asthma appear to decrease at least in Western countries; but there are significant variations between populations. The aim of this study is to describe the trends in deaths due to asthma between 1993 and 2007 in our community and to analyze any factors associated with this. METHODS: All cases, which had asthma as a cause of death, were included in the study. The mortality data were obtained from the official death certificate from the Deaths Register. Mortality relative risk, based on the calendar year, sociodemographic variables (age and gender), seasons, and days of the week, were estimated using a Poisson generalized linear model with a log-link. RESULTS: In the 15 years of the study period, a total of 1180 people had died due to asthma, mainly in winter (34.5%), women (64.5%), and advanced age (65 years or above; 84.6%). A tendency of decreased mortality in all age groups was observed during the period of the study, which was significant in both the sexes in the 35- to 64-year-old age group, and in males above 65 years. CONCLUSION: Death due to asthma is changing favorably in our community, with a tendency to decrease in the past few years. Advanced age, being female, and the winter period are associated with a higher mortality rate due to asthma.

A randomized trial comparing the efficacy and safety of imiglucerase (Cerezyme) infusions every 4 weeks versus every 2 weeks in the maintenance therapy of adult patients with Gaucher disease type 1.

Imiglucerase (Cerezyme) has been the standard of care for treatment of Gaucher disease, a lysosomal storage disorder resulting from deficiency of glucocerebrosidase, since its approval in 1994. Infusions are typically given once every 2 weeks. However, many patients have expressed a desire for less frequent infusions as a matter of convenience. This clinical study assessed the safety and efficacy of intravenous imiglucerase infused once every 4 weeks (Q4) compared to once every 2 weeks (Q2) at the same total monthly dose in adult patients with clinically stable Gaucher disease type 1 (GD1). This was a 24-month, open-label, randomized, Phase 4, dose-frequency study conducted in 25 centers worldwide. Patients receiving imiglucerase were randomized to receive their monthly dose biweekly (n=33) or every 4 weeks (n=62). Changes from baseline in hemoglobin, platelets, liver and spleen volumes, bone crisis, and bone disease comprised a predefined composite endpoint; achievement or maintenance of established Gaucher disease therapeutic goals comprised a secondary endpoint. Sixty-three percent of Q4- and 81% of Q2-treated patients met the composite endpoint at Month 24; 89% of Q4- and 100% of Q2-treated patients met the therapeutic goals-based endpoint. The frequency of related adverse events was comparable between treatment groups. This study suggests that with comprehensive monitoring, a Q4 imiglucerase infusion regimen may be a safe and effective treatment option for the majority of clinically stable adult patients with GD1 but may not be appropriate for all GD1 patients. Continued monitoring in patients treated with Q4 dosing is required to assess long-term effectiveness.

Clinical features of chromosome 22q11.2 microdeletion syndrome in 208 Chilean patients.

Patients with chromosome 22q11 deletion syndrome exhibit significant phenotypic variability. Epidemiologic data suggest a higher incidence in Hispanics, but limited clinical information is available from Latin-American patients. We describe the clinical features of Chilean patients with 22q11 deletion syndrome and compare their findings with those reported in large European, Japanese and US series. Data were obtained from 208 patients from five medical centers. Mean age at diagnosis was 5.2 years, with a median of 2.3 years. Congenital heart defects were present in 59.6%, lower than other large series that averaged 75.8%. Palate abnormalities were present in 79%, higher than previous reports averaging 56%. Patients with congenital heart disease were diagnosed earlier (median 0.3 years of age) than those without heart defects (median 5.6 years) and had greater mortality attributable to the syndrome (9.8% vs 2.4%, respectively). The differences in frequencies of major anomalies may be due to growing awareness of more subtle manifestations of the syndrome, differences in clinical ascertainment or the presence of modifier factors. These observations provide additional data useful for patient counseling and for the proposal of health care guidelines.

Hydration status, drug interactions, and determinants in a Spanish elderly population: a pilot study.

Proper hydration is essential to maintain optimal health and well-being at all stages of life, especially for the elderly. Side effects of certain drugs that affect hydration status may compromise the health of the ancients, who also constitute the most vulnerable group. No studies have been carried out, to our knowledge, at the intersection of drugs and hydration status. Our study aimed to evaluate the effects of chronic use of certain drugs (diuretics, corticoids and metformin) in the hydration status of the elderly. Results were obtained from a cross-sectional study with 96 volunteers (65-93 years) selected based on their pharmacological treatment. It included a validated food and drink frequency questionnaire and water removal, dehydration signs and symptoms assessment and urine analysis. All data were analysed by age and sex. Water balance decreased with advanced age, especially in men's group. Results were confirmed by means of the evaluation of dehydration signs and symptoms and colorimetric and chemical analysis of urine. Correlations between consumption of corticoids and hydration status were found, with different signs depending on the administration route (Rho = 0.522 and Rho = - 0,522 for oral and pulmonary corticoids, respectively). Furthermore, correlations between diuretics (Rho = - 0.343, p < 0.05) and metformin (Rho = - 0.802, p < 0.01) consumption and different urine markers were determined. In conclusion, the predominant dehydration state of the volunteers of the study is affected by drugs consumption and their route of administration. Hence, there is an urgent need for monitorization of hydration status based on drugs consumption.

Epigenetics as a mechanism linking developmental exposures to long-term toxicity.

A variety of experimental and epidemiological studies lend support to the Developmental Origin of Health and Disease (DOHaD) concept. Yet, the actual mechanisms accounting for mid- and long-term effects of early-life exposures remain unclear. Epigenetic alterations such as changes in DNA methylation, histone modifications and the expression of certain RNAs have been suggested as possible mediators of long-term health effects of environmental stressors. This report captures discussions and conclusions debated during the last Prenatal Programming and Toxicity meeting held in Japan. Its first aim is to propose a number of criteria that are critical to support the primary contribution of epigenetics in DOHaD and intergenerational transmission of environmental stressors effects. The main criteria are the full characterization of the stressors, the actual window of exposure, the target tissue and function, the specificity of the epigenetic changes and the biological plausibility of the linkage between those changes and health outcomes. The second aim is to discuss long-term effects of a number of stressors such as smoking, air pollution and endocrine disruptors in order to identify the arguments supporting the involvement of an epigenetic mechanism. Based on the developed criteria, missing evidence and suggestions for future research will be identified. The third aim is to critically analyze the evidence supporting the involvement of epigenetic mechanisms in intergenerational and transgenerational effects of environmental exposure and to particularly discuss the role of placenta and sperm. While the article is not a systematic review and is not meant to be exhaustive, it critically assesses the contribution of epigenetics in the long-term effects of environmental exposures as well as provides insight for future research.

North African-Mediterranean HLA genetic contribution in a population of the kidney transplant waiting list patients of Canary origin (Gran Canaria).

The peopling of the Canary Islands has been widely debated. The mitochondrial DNA and Y-chromosome data support the idea of a Berber genetic origin coming from the North of Africa (maternal) and a later contribution of the Spanish invaders (paternal). The frequencies of the HLA class II alleles from the Tenerife Island (another Canary Island) have previously been published, postulating a Berber and Atlantic/Iberian contributions to the current population. The HLA class I and class II allele frequencies, haplotype frequencies and phylogenetic comparisons were performed in 215 unrelated individuals from Gran Canaria Island (belonging to the kidney transplant waiting list), with at least three generations of ancestors from Canary Islands, in order to study the different ethnical HLA contributions to the genetic background of the Canary Islanders. Results showed the presence of a compound HLA haplotype of putative Phoenician-Berber origin, A*33:01-C*08:02-B*14:02-DRB1*03:01-DQB1*02:01, likely coming from the combination of haplotypes A*30:02-C*05:01-B*18:01-DRB1*03:01-DQB1*02:01 and A*33:01-C*08:02-B*14:02-DRB1*01:02-DQB1*05:01 of North African (probably Berber) and West Asian Mediterranean (probably Phoenician) origins, respectively. The latter haplotypes and others from the same origin (Berber/Phoenician) are also present in the population studied. Besides, other contributions from the North of Europe, North England-Iberian (Atlantic contribution), and Western Europe/Mediterraneans (Spanish colonization) are also discussed. These data conclude that the current genetic background of the Canary Islands inhabitants has been generated over the years by different ways with an original Phoenician-Berber substrate and several genetic contributions generated in different invasions.

Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection.

Although recombinant adenoviruses are attractive vectors for gene transfer to airway epithelia, they have proven to be relatively inefficient. To investigate the mechanisms of adenovirus-mediated gene transfer to airway epithelia, we examined the role of adenovirus fiber and penton base, the two proteins involved in attachment to and entry of virus into the cell. We used human airway epithelia grown under conditions that allow differentiation and development of a ciliated apical surface that closely resembles the in vivo condition. We found that addition of fiber protein inhibited virus binding and vector-mediated gene transfer to immature airway epithelia, as well as to primary cultures of rat hepatocytes and HeLa cells. However, fiber protein had no effect on vector binding and gene transfer to ciliated airway epithelia. We obtained similar results with addition of penton base protein: the protein inhibited gene transfer to immature epithelia, whereas there was no effect with ciliated epithelia. Moreover, infection was not attenuated with an adenovirus containing a mutation in penton base that prevents the interaction with cell surface integrins. These data suggest that the receptors required for efficient infection by adenovirus are either not present or not available on the apical surface of ciliated human airway epithelia. The results explain the reason for inefficient gene transfer and suggest approaches for improvement.

A mouse model for the delta F508 allele of cystic fibrosis.

The most common cause of cystic fibrosis is a mutation that deletes phenylalanine 508 in cystic fibrosis transmembrane conductance regulator (CFTR). The delta F508 protein is misprocessed and degraded rather than traveling to the apical membrane. We used a novel strategy to introduce the delta F508 mutation into the mouse CFTR gene. Affected epithelia from homozygous delta F508 mice lacked CFTR in the apical membrane and were Cl-impermeable. These abnormalities are the same as those observed in patients with delta F508 and suggest that these mice have the same cellular defect. 40% of homozygous delta F508 animals survived into adulthood and displayed several abnormalities found in human disease and in CFTR null mice. These animals should provide an excellent model to investigate pathogenesis and to examine therapies directed at correcting the delta F508 defect.

Correlation of MR imaging and MR spectroscopy findings with cognitive impairment in mucopolysaccharidosis II.

BACKGROUND AND PURPOSE: There are no reliable markers to predict neurologic outcome of patients with mucopolysaccharidosis (MPS) II. We hypothesized that brain MR imaging and MR spectroscopy are useful in depicting features related to cognitive impairment (CI) in MPS II. MATERIALS AND METHODS: Nineteen male patients with MPS II were included in this study. They were evaluated through intelligence/developmental tests to be classified in 2 groups: patients with CI (group A) or patients without CI (group B). Brain MR imaging evaluated white matter (WM) lesions, hydrocephalus, and brain atrophy. Voxels from MR spectroscopy (point-resolved spectroscopy TE 30 ms) were positioned in the WM of the deep right frontal lobe and at the gray matter (GM) in the posterior occipital cortex across the midline. Comparison of MR imaging and MR spectroscopy findings between these 2 groups and a control group was performed. RESULTS: The mean age of the patients was 9.6 years (group A, 7.08 years old, 12 patients; group B, 14 years old, 7 patients; P = .076). Brain atrophy and hydrocephalus were more frequently found in group A patients (P=.006 and P=.029, respectively); these patients also presented more severe WM lesions than patients from group B (P=.022). Patients from group A also had a higher myo-inositol (mIns)/creatine (Cr) ratio in the GM (P=.046) and in the WM (P=.032). The choline/Cr and N-acetylaspartate/Cr ratios were similar in both groups. CONCLUSIONS: Our study showed that severe WM lesions, brain atrophy, hydrocephalus, and elevated mIns/Cr were more common in patients with MPS II and with CI.

Constitutive activation of the aromatic hydrocarbon receptor.

The ligand-activated aromatic hydrocarbon receptor (AHR) dimerizes with the AHR nuclear translocator (ARNT) to form a functional complex that transactivates expression of the cytochrome P-450 CYP1A1 gene and other genes in the dioxin-inducible [Ah] gene battery. Previous work from this laboratory has shown that the activity of the CYP1A1 enzyme negatively regulates this process. To study the relationship between CYP1A1 activity and Ah receptor activation we used CYP1A1-deficient mouse hepatoma c37 cells and CYP1A1- and AHR-deficient African green monkey kidney CV-1 cells. Using gel mobility shift and luciferase reporter gene expression assays, we found that c37 cells that had not been exposed to exogenous Ah receptor ligands already contained transcriptionally active AHR-ARNT complexes, a finding that we also observed in wild-type Hepa-1 cells treated with Ellipticine, a CYP1A1 inhibitor. In CV-1 cells, transient expression of AHR and ARNT leads to high levels of AHR-ARNT-dependent luciferase gene expression even in the absence of an agonist. Using a green fluorescent protein-tagged AHR, we showed that elevated reporter gene expression correlates with constitutive nuclear localization of the AHR. Transcriptional activation of the luciferase reporter gene observed in CV-1 cells is significantly decreased by (i) expression of a functional CYP1A1 enzyme, (ii) competition with chimeric or truncated AHR proteins containing the AHR ligand-binding domain, and (iii) treatment with the AHR antagonist alpha-naphthoflavone. These results suggest that a CYP1A1 substrate, which accumulates in cells lacking CYP1A1 enzymatic activity, is an AHR ligand responsible for endogenous activation of the Ah receptor.

Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation.

Transcriptional activation of the murine Cyp1a-1 (cytochrome P(1)450) gene by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (dioxin) requires the aromatic hydrocarbon (Ah) receptor and the interaction of an inducer-receptor complex with one or more of the Ah-responsive elements (AhREs) located about 1 kb upstream from the transcriptional initiation site. We find that treatment of mouse hepatoma Hepa-1 cells with 2-aminopurine, an inhibitor of protein kinase activity, inhibits CYP1A1 mRNA induction by TCDD as well as the concomitant increase in CYP1A1 enzyme activity. Formation of DNA-protein complexes between the Ah receptor and its AhRE target is also inhibited by 2-aminopurine, as determined by gel mobility shift assays. Phosphorylation is required for the formation of Ah receptor-specific complexes, since in vitro dephosphorylation of nuclear extracts from TCDD-treated Hepa-1 cells abolishes the capacity of the Ah receptor to form specific complexes with its cognate AhRE sequences. To determine whether any one of several known protein kinases was involved in the transcriptional regulation of the Cyp1a-1 gene, we treated Hepa-1 cells with nine other protein kinase inhibitors prior to induction with TCDD; nuclear extracts from these cells were analyzed for their capacity to form specific DNA-protein complexes. Only extracts from cells treated with staurosporine, a protein kinase C inhibitor, were unable to form these complexes. In addition, staurosporine completely inhibited CYP1A1 mRNA induction by TCDD. Depletion of protein kinase C by prolonged treatment with phorbol ester led to the complete suppression of CYP1A1 mRNA induction by TCDD. We conclude that (i) phosphorylation is necessary for the formation of a transcriptional complex and for transcriptional activation of the Cyp1a-1 gene; (ii) the phosphorylation site(s) exists on at least one of the proteins constituting the transcriptional complex, possibly the Ah receptor itself; and (iii) the enzyme responsible for the phosphorylation is likely to be protein kinase C.

Restoration of retinoblastoma mediated signaling to Cdk2 results in cell cycle arrest.

Phosphorylation/inactivation of RB is typically required for cell cycle progression. However, we have identified a tumor cell line, C33A, which progresses through the cell cycle in the presence of an active allele of RB (PSM-RB). To determine how C33A cells evade RB-mediated arrest, we compared RB signaling to downstream effectors in this resistant cell line to that of the RB-sensitive SAOS-2 cell line. Although introduction of PSM-RB repressed E2F-mediated transcription in both C33A and SAOS-2 cells, PSM-RB failed to repress Cyclin A promoter activity in C33A. Ectopic expression of PSM-RB in SAOS-2 cells resulted in a decrease in both Cyclin A and Cdk2 protein levels without affecting Cyclin E or Cdk4. In contrast, over-expression of PSM-RB in C33A cells did not alter endogenous Cyclin A, Cyclin E, or Cdk2 protein levels or impact Cdk2 kinase activity, indicating that signaling from RB to down-stream targets is abrogated in this cell line. The importance of Cdk2 activity was demonstrated by p27Kip1, which attenuated Cdk2 activity and inhibited cell cycle progression in C33A cells. Since RB signaling to Cdk2 is disrupted in these tumor cells, we co-expressed two proteins that cooperate with RB in transcriptional repression, AHR and BRG-1, in an attempt to correct this signaling dysfunction. Co-expression of AHR/BRG-1 with PSM-RB attenuated Cyclin A and Cdk2 expression as well as Cdk2-associated kinase activity, resulting in cell cycle inhibition of C33A cells. Importantly, ectopic expression of Cyclin A was able to reverse the arrest mediated by co-expression of AHR/BRG-1 with PSM-RB. These results indicate that down-regulation of Cdk2 activity is requisite for RB-mediated cell cycle arrest. Thus, this study reveals a new mechanism through which tumor cells evade anti-proliferative signals, and provides insight into how RB-signaling is mediated.

The murine Cyp1a-1 gene negatively regulates its own transcription and that of other members of the aromatic hydrocarbon-responsive [Ah] gene battery.

Transcripts of the murine CYP1A1 (cytochrome P1450) mRNA are markedly elevated in mutant hepatoma cell lines that contain missense mutations in the Cyp1a-1 structural gene. This putative derepression extends to other genes in the [Ah] battery. To test whether the Cyp1a-1 gene product is involved in a mechanism of feedback regulation of transcription, we introduced expression plasmids carrying the murine wild-type Cyp1a-1 cDNA into the mutant hepatoma cells. Measurements of steady-state mRNA levels and of transcriptional rates in the transfectants reveal that expression of a functional, exogenous CYP1A1 protein is sufficient to restore the repression of the endogenous gene, as well as restore the inducibility by dioxin, and that this effect takes place primarily at the level of transcription. Similar experiments with expression plasmids that carry the human CYP1A2 cDNA indicate that the CYP1A2 protein (cytochrome P3450) can also function as a transcriptional repressor. In addition, we find that expression of the Nmo-1 [NAD(P)H:menadione oxidoreductase] gene, a third member of the [Ah] gene battery, is also repressed by the exogenous expression of either Cyp1a-1 or CYP1A2 cDNA. These results indicate that the gene product of either member of the mammalian CYP1 family has a previously unrecognized transcriptional regulatory function, which is likely to be exerted by modification of preexisting trans-acting factors. This function may help bring about a fast reprogramming of gene expression, as might be needed during detoxification of toxic foreign chemicals.

Cadmium, lead, and zinc mobility and plant uptake in a mine soil amended with sugarcane straw biochar.

Accumulation of heavy metals in unconsolidated soils can prove toxic to proximal environments, if measures are not taken to stabilize soils. One way to minimize the toxicity of metals in soils is the use of materials capable of immobilizing these contaminants by sorption. Biochar (BC) can retain large amounts of heavy metals due to, among other characteristics, its large surface area. In the current experiment, sugarcane-straw-derived biochar, produced at 700 °C, was applied to a heavy-metal-contaminated mine soil at 1.5, 3.0, and 5.0% (w/w). Jack bean and Mucuna aterrima were grown in pots containing a mine contaminated soil and soil mixed with BC. Pore water was sampled to assess the effects of biochar on zinc solubility, while soils were analyzed by DTPA extraction to confirm available metal concentrations. The application of BC decreased the available concentrations of Cd, Pb, and Zn in the mine contaminated soil leading to a consistent reduction in the concentration of Zn in the pore water. Amendment with BC reduced plant uptake of Cd, Pb, and Zn with the jack bean uptaking higher amounts of Cd and Pb than M. aterrima. This study indicates that biochar application during mine soil remediation could reduce plant concentrations of heavy metals. Coupled with this, symptoms of heavy metal toxicity were absent only in plants growing in pots amended with biochar. The reduction in metal bioavailability and other modifications to the substrate induced by the application of biochar may be beneficial to the establishment of a green cover on top of mine soil to aid remediation and reduce risks.

Human AH locus polymorphism and cancer: inducibility of CYP1A1 and other genes by combustion products and dioxin.

The polymorphism of mammalian aromatic hydrocarbon (Ah) responsiveness appears to be correlated with genetic differences in risk of bronchogenic carcinoma caused by cigarette smoking. The human polymorphism has been uncovered, largely as the result of corresponding genetic differences characterized first in the mouse. The murine Ah locus has been defined as the gene encoding the aromatic hydrocarbon-responsive (Ah) receptor, responsible for the inducibility of a battery of at least six genes, two of which encode P450 enzymes. The high-affinity receptor and, hence, more highly induced levels of P450, can result in greater concentrations of polycyclic aromatic reactive intermediates that form DNA adducts and, ultimately, mutation fixation (tumour initiation). The Ah receptor is also likely to participate in growth and differentiation signal transduction pathways (tumour promotion). Positive and negative control regions flanking the murine Cyp 1a-1 and human CYP1A1 (cytochrome P(1)450) genes have been identified. A DNA motif approximately 1 kb upstream of the transcription start site appears to affect the translatability of the CYP1A1 mRNA and activity of the enzyme. Expression of the CYP1A1 or CYP1A2 enzyme in mouse hepatoma Hepa-1 cells lacking endogenous CYP1A1 activity represses constitutive transcription of not only the endogenous Cyp1a-1 gene but other genes in the dioxin-inducible [Ah] battery. Human polymorphisms involving a Msp I site 450 bp downstream from the last CYP1A1 exon have been described in Japan, the Eastern Mediterranean, Norway and the USA. The '1.9 allele' is associated with an increased incidence of Kreyberg Type I bronchogenic carcinomas in Japan and has recently been correlated with a valine-to-isoleucine substitution at position 462 in the haeme-binding region. This allele is about 3 times more frequent in Japan than in Caucasians of Norway and the USA, in which no correlation has been found between this allele and lung cancer. More work is needed to clarify these findings. Isolation and sequencing of the human Ah receptor cDNA, and the subsequent screening of populations for polymorphisms, hold great promise for predicting interindividual risk of cancer caused by smoking and other environmental pollutants.

Human Ah receptor (AHR) gene: localization to 7p15 and suggestive correlation of polymorphism with CYP1A1 inducibility.

The mammalian aromatic hydrocarbon receptor (AHR) is a ubiquitous ligand-activated transcription factor. AHR ligands include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), benzo[a]pyrene, and polychlorinated and polybrominated biphenyls; the endogenous ligand is not yet known. Following ligand binding, the AHR transcriptionally activates genes encoding drug-metabolizing enzymes important in both the metabolic potentiation of substrates to genotoxic reactive intermediates and ultimate carcinogens, and the detoxification of toxic or carcinogenic drugs and other environmental pollutants. AHR-mediated gene expression is also involved in many critical life processes (e.g. cell type-specific differentiation, cell division, apoptosis) by signal transduction mechanisms. Similar to mice, human populations exhibit a > 20-fold range of the CYP1A1 inducibility/AHR affinity phenotype. In the present study, we localized the human AHR gene to chromosome 7p15, using fluorescence in situ hybridization (FISH). Performing linkage analysis in a three-generation family, we show with good probability that the high CYP1A1 inducibility phenotype segregates with the 7p15 region. Sequencing 93 nt (31 amino acids) of the human AHR gene's exon 9, which is the region correlated with the mouse A375V polymorphism responsible for the major portion of high vs low CYP1A1 inducibility/AHR affinity, we found no nucleotide differences; Val-381 was present in all five individuals examined (four related and one unrelated), two of whom show "high' and three of whom show "low' CYP1A1 inducibility. These data indicate that the "high' and "low' CYP1A1 inducibility trait, in the population studied, cannot be explained by a difference among these 31 amino acids in exon 9 of the AHR gene.

Mouse dioxin-inducible NAD(P)H: menadione oxidoreductase: NMO1 cDNA sequence and genetic differences in mRNA levels.

We have cloned and sequenced the mouse NMO1 cDNA, which encodes the NAD(P)H:menadione oxidoreductase [also called NAD(P)H:(quinone acceptor) oxidoreductase; quinone reductase; azo dye reductase; DT diaphorase; EC 1.6.99.2]. The cDNA is 1528 bp in length excluding the poly(A+) tail, and has 5' and 3' nontranslated regions of 108 bp and 595 bp, respectively. The deduced protein contains 274 amino acids, including the first methionine (M(r) = 30,959). The mouse NMO1 protein is: 94% similar to the rat NMO1 and 86.5% to the human NMO1 proteins; 49.3% identical to the human NQO2 protein; and < 20% similar to several dozen other proteins in the quinone oxidoreductase superfamily. Southern hybridization analysis of mouse DNA reveals that the Nmo1 gene is likely to span less than a total of 20 kb. The Nmo1 gene is highly inducible by 2,3,7,8,-tetrachlorodibenzo-p-dioxin (dioxin; TCDD) in mouse liver and mouse cell cultures. TCDD inducibility of NMO1 is detectable at 12 and 18 days of gestation, but markedly elevated at 1-3 weeks post partum as compared with the 6- and 12-week-old mouse. NMO1 mRNA levels are strikingly elevated in the untreated mouse hepatoma Hepa-1c1c7 mutant line c37 lacking CYP1A1 (aryl hydrocarbon hydroxylase) activity, and in the untreated 14CoS/14CoS mouse cell line having an 'oxidative stress response' caused by homozygous deletion of about 3800 kb on chromosome 7. Previous work and the data in this report show that the murine Nmo1 gene is regulated by three distinct mechanisms: CYP1A1 metabolism-dependent repression, Ah receptor-mediated induction by TCDD, and activation by the chromosome 7-mediated oxidative stress response.