The role of aristolochic acid in the etiology of Balkan endemic nephropathy (BEN) and associated upper-tract urothelial carcinoma (UTUC) has been recently confirmed. The aim of this study was to determine apoptosis-related marker(s) specific for BEN-associated UTUC. Present investigation included 105 patients with UTUC, 44 from BEN region and 61 control tumors. Altered expression of Survivin was more often present in BEN UTUC with high grade and solid growth (P < 0.005; P < 0.05) than in control tumors. Significantly lower expression of proapoptotic marker Bax was found in BEN tumors with high grade, high stage, necrosis, and without metaplastic change (P < 0.05; 0.05; 0.05; 0.05) compared to control tumors with the same features. Group (BEN-related/control), stage, growth pattern, and caspase 3 activity were significantly associated with the expression of Bax (P = 0.002, 0.034, 0.047, 0.028, resp.,). This investigation identifies Bax as specific marker of BEN-associated UTUC. Decrease of pro-apoptotic protein Bax together with alteration of Survivin may be indicative for specific disturbances of intrinsic apoptotic pathway in UTUC arising in endemic areas.
Balkan endemic nephropathy; upper tract urothelial carcinoma; apoptosis; Survivin; Bcl-2; Bax; Fas; Caspase 3
The etiology of Balkan Endemic Nephropathy, (BEN), a tubulointerstitial kidney disease, is unknown. Although this disease is endemic in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia, similar manifestations are reported to occur in other regions, for instance Tunisia and Sri Lanka. A number of explanations have been stated including lignites, aristolochic acid, ochratoxin A, metals, and metalloids. Etiologic claims are often based on one or a few studies without sound scientific evidence. In this systematic study, we tested whether exposures to metals (cadmium and lead) and metalloids (arsenic and selenium) are related to Balkan Endemic Nephropathy.
In 2003/04 we recruited 102 adults whose parents had BEN and who resided in one of three communities (Vratza, Bistretz, or Beli Izvor, Bulgaria). A control group comprised of 99 adults having non-BEN hospitalized parents was enrolled in the study during the same time. We conducted face-to-face interviews, ultrasound kidney measurements, and determined kidney function in two consecutive investigations (2003/04 and 2004/05). Metals and metalloids were measured in urine and blood samples. To assess the agreement between these consecutive measurements, we calculated intraclass correlation coefficients. Repeated measurement data were analyzed using mixed models.
We found that cadmium and arsenic were associated with neither kidney size nor function. Lead had a significant but negligible effect on creatinine clearance. Selenium showed a weak but significant negative association with two of the four kidney parameters, namely creatinine clearance and β2-microglobulin. It was positively related to kidney length. These associations were not restricted to the offspring of BEN patients. Adding credence to these findings are reports showing comparable kidney effects in animals exposed to selenium.
The findings of this 2-year follow-up study indicate that metals and metalloids do not play a role in the etiology of Balkan Endemic Nephropathy. Against the assumption in the literature, selenium was not protective but a risk factor. Since comparable associations were observed in animals, future studies are needed to explore whether selenium may have adverse renal effects in humans.
We evaluated the role of exposure analysis in assessing whether ochratoxin A aristolochic acid are the agents responsible for causing Balkan endemic nephropathy. We constructed a framework for exposure analysis using the lessons learned from the study of endemic goiter within the context of an accepted general model. We used this framework to develop an exposure analysis model for Balkan endemic nephropathy, evaluated previous findings from the literature on ochratoxin A and aristolochic acid in the context of this model, discussed the strength of evidence for each, and proposed approaches to address critical outstanding questions. The pathway for exposure to ochratoxin A is well defined and there is evidence that humans have ingested ochratoxin A. Factors causing differential exposure to ochratoxin A and how ochratoxin A is implicated in Balkan endemic nephropathy are not defined. Although there is evidence of human exposure to aristolochic acid and that its effects are consistent with Balkan endemic nephropathy, a pathway for exposure to aristolochic acid has been suggested but not demonstrated. Factors causing differential exposure to aristolochic acid are not known. Exposure analysis results suggest that neither ochratoxin A nor aristolochic acid can be firmly linked to Balkan endemic nephropathy. However, this approach suggests future research directions that could provide critical evidence on exposure, which when linked with findings from the health sciences, may be able to demonstrate the cause of this disease and provide a basis for effective public health intervention strategies. One of the key unknowns for both agents is how differential exposure can occur.
Upper tract urothelial carcinoma (UTUC) associated with Balkan endemic nephropathy (BEN) is characterized by a number of aberrations in cell-cycle regulation and apoptosis. The aim of this study was to detect angiogenesis-related marker(s) specific for BEN UTUC, and to examine the influence of HIF 1α upon angiogenesis and apoptosis in UTUC. Present investigation included 110 patients with UTUC, 50 from BEN region and 60 control tumors. Altered expression of VEGFR1 was more often present in control UTUC than in BEN tumors (p<0.005). It was associated with high grade, low and high stage, solid growth, and metaplastic change of control UTUC. Microvessel density assessed by CD31 (MVD CD31) was significantly higher in UTUC with lymphovascular invasion (p<0.05), and in BEN tumors with papillary growth (p<0.05). Discriminant analysis indicated that BEN and control tumors do not differ significantly in expression of angiogenesis related markers. The most important discriminant variable that determined control UTUC was expression of VEGFR1 (p=0.002). HIF 1α in UTUC significantly correlated with the low stage, papillary growth and expression of Bcl-2, Caspase-3 index, and MVD CD34 (p<0.001; 0.0005; 0.01; 0.005; 0.01, respectively). HIF-1α may be helpful marker in evaluation of UTUC, especially when combined with angiogenesis and apoptosis.
Balkan endemic nephropathy; upper tract urothelial carcinoma; angiogenesis; VEGF; VEGFR1; VEGFR2; CD31; CD34; HIF 1α; apoptosis
Balkan endemic nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from non-endemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by 32P-post-labeling, the adduct was confirmed by mass spectroscopy, and TP53 mutations in tumor tissues were identified by chip-sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in non-endemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.
Balkan endemic nephropathy (BEN) is a chronic kidney disease that affects persons living in the Balkans. Despite the unique geographical specificity of this disease, its etiology has remained unclear. Even if a positive family history of BEN has been identified, it is still uncertain how the disease develops in offspring. In this paper, we examine clinical mechanisms related to the onset of BEN in individuals who have a parental history of BEN to identify early detection of the disease and formulate interventions. We conducted a 5-year prospective study, using markers in years one and three to predict new cases of BEN in year five. New cases of BEN were defined based on three criteria: parental history of BEN, reduced kidney size, and reduced kidney function. Incident cases were divided into (1) probable, (2) definite, and (3) combined labeled total incidence. We evaluated parental history in relation to BEN and tested the potentially intervening effects of kidney length, kidney cortex width, β2-microglobulin, C-reactive protein, and creatinine clearance, using path analyses. The findings of the path analyses suggested that parental history of BEN had both direct and indirect effects. The direct effect was significant for all three modes of parental history (biparental, maternal, and paternal; odds ratios 71.5, 52.3, and 50.1, respectively). The indirect effects of maternal BEN acted via kidney length and creatinine clearance. Biparental BEN was mediated by (1) kidney length and creatinine clearance, and (2) creatinine clearance alone. Paternal BEN had three indirect effects: (1) through kidney length and creatinine clearance, (2) via kidney cortex width and creatinine clearance, and (3) via kidney cortex width only. In conclusion, a family history of BEN led to reduced kidney length and cortex width, and a decline in creatinine clearance, which in turn predicted the onset of BEN.
Balkan endemic nephropathy; incidence of BEN; parental history of BEN; kidney size; β2-microglobulin; creatinine clearance
Aristolochic acids are nephrotoxic and carcinogenic natural products that have been implicated both in the endemic nephropathy in the Balkans region and ailments caused by ingestion of herbal remedies. Aristolochic acids are metabolized to active intermediates that bind to DNA. In this study, reduction of aristolochic acid I with zinc/acetic acid afforded a new product which was characterized as 9-methoxy-7-methyl-2H-1,3-oxazolo[5′,4′-10,9]phenanthro[3,4-d]1,3-dioxolane-5-carboxylic acid, designated as aristoxazol, along with the expected aristolactam I. This new compound is a condensation product of aristolochic acid and acetic acid that may be related to the aristolochic acid-DNA-adducts. The proposed mechanism of formation of aristoxazol involves nucleophilic attack of acetic acid on the nitrenium ion of aristolochic acid I. On the basis of these studies a route to the metabolic activation of aristolochic acids and formation of adducts with DNA in in vitro systems is proposed and discussed.
Previous studies have linked smaller kidney dimensions to increased blood pressure. However, patients with Balkan Endemic Nephropathy (BEN), whose kidneys shrink during the course of the disease, do not manifest increased blood pressure. The authors evaluated the relationship between kidney cortex width, kidney length, and blood pressure in the offspring of BEN patients and controls.
102 offspring of BEN patients and 99 control offspring of non-BEN hospital patients in the Vratza District, Bulgaria, were enrolled in a prospective study and examined twice (2003/04 and 2004/05). Kidney dimensions were determined using ultrasound, blood pressure was measured, and medical information was collected. The parental disease of BEN was categorized into three groups: mother, father, or both parents. Repeated measurements were analyzed with mixed regression models.
In all participants, a decrease in minimal kidney cortex width of 1 mm was related to an increase in systolic blood pressure of 1.4 mm Hg (p = 0.005). There was no association between kidney length and blood pressure. A maternal history of BEN was associated with an increase in systolic blood pressure of 6.7 mm Hg (p = 0.03); paternal BEN, +3.2 mm Hg (p = 0.35); or both parents affected, +9.9 mm Hg (p = 0.002). There was a similar relation of kidney cortex width and parental history of BEN with pulse pressure; however, no association with diastolic blood pressure was found.
In BEN and control offspring, a smaller kidney cortex width predisposed to higher blood pressure. Unexpectedly, a maternal history of BEN was associated with average increased systolic blood pressure in offspring.
Despite the characteristic extensive tubulointerstitial fibrosis, Balkan Endemic Nephropathy (BEN) is usually considered a non-inflammatory disease.
We examined a marker of inflammation, C-reactive protein (CRP), in the offspring of patients with BEN, a population at risk for BEN, prior to development of established disease to determine if an inflammatory process could be identified in the early stages of the disease. In 2003/04, 102 adult offspring whose parents had BEN and a control group of 99 adult offspring of non-BEN patients were enrolled in this prospective study. This cohort was re-examined yearly for four consecutive years. Levels of serum CRP were measured in years 3 and 4 and compared between groups. The data were analyzed with mixed models.
Compared to controls, offspring of BEN parents had statistically higher CRP levels in two consecutive years, suggestive of early inflammatory reactivity. Whenever the mother was affected by BEN (both parents, or mother only), serum CRP was significantly increased, but not if only the father had BEN. CRP was inversely related to kidney cortex width but not to markers or renal function.
Early stages of BEN may involve inflammatory processes. The observation of a maternal involvement supports the concept of fetal programming, which has been implicated in the pathogenesis of other chronic kidney diseases.
Ochratoxin A (OTA) is a mycotoxin naturally occurring in different foods. OTA is arguably a risk factor for Balkan endemic nephropathy (BEN). The aims of this study are to (1) test the OTA-BEN association in BEN-groups and controls and (2) determine whether urine β2-microglobulin, a marker of impaired ability of the kidneys to re-absorb, is related to OTA. BEN patients had significantly higher OTA serum levels. Within the offspring, OTA was significantly related to higher β2-microglobulin excretion. OTA (2005/2006) was related to a higher incidence of BEN after 2008, providing further evidence that OTA is a risk factor for BEN.
BEN; ochratoxin A; β2-microglobulin
1H NMR spectroscopy of urine has been applied to exploring metabolomic differences between people diagnosed with Balkan endemic nephropathy (BEN), and treated by haemodialysis, and those without overt renal disease in Romania and Bulgaria. Convenience sampling was made from patients receiving haemodialysis in hospital and healthy controls in their village. Principal component analysis clustered healthy controls from both countries together. Bulgarian BEN patients clustered separately from controls, though in the same space. However, Romanian BEN patients not only also clustered away from controls but also clustered separately from the BEN patients in Bulgaria. Notably, the urinary metabolomic data of two people sampled as Romanian controls clustered within the Romanian BEN group. One of these had been suspected of incipient symptoms of BEN at the time of selection as a ‘healthy’ control. This implies, at first sight, that metabolomic analysis can be predictive of impending morbidity before conventional criteria can diagnose BEN. Separate clustering of BEN patients from Romania and Bulgaria could indicate difference in aetiology of this particular silent renal atrophy in different geographic foci across the Balkans.
Balkan nephropathy; metabolomics; urinalysis; haemodialysis; ochratoxin A; aristolochic acid
Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy, which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA may also cause a similar type of kidney fibrosis with malignant transformation of the urothelium, the Balkan endemic nephropathy. Understanding which enzymes are involved in AA activation and/or detoxication is important in the assessment of a susceptibility to this carcinogen. The most important human enzymes activating AA by simple nitroreduction in vitro are hepatic and renal cytosolic NAD(P)H:quinone oxidoreductase, hepatic microsomal cytochrome P450 1A2 and renal microsomal NADPH:cytcohrome P450 reductase, besides cyclooxygenase, which is highly expressed in urothelial tissue. Despite extensive research, contribution of most of these enzymes to the development of these diseases is still unknown. Hepatic cytochromes P450 were found to detoxicate AA in mice, and thereby protect the kidney from injury. However, which of cytochromes P450 are the most important in this process both in animal models and in humans have not been entirely resolved as yet. In addition, the relative contribution of enzymes found to activate AA to species responsible for induction of urothelial cancer in humans remains still to be resolved.
Aristolochic acid; metabolism; Aristolochic acid- and Balkan endemic-nephropathy; renal injury; tumor induction
The Pliocene lignite hypothesis is an environmental hypothesis that has been proposed to explain the etiology of Balkan endemic nephropathy (BEN). Aqueous leaching experiments were conducted on a variety of coal samples in order to simulate groundwater leaching of organic compounds, and to further test the role of the Pliocene lignite hypothesis in the etiology of BEN. Experiments were performed on lignite coal samples from endemic BEN areas in Romania and Serbia, and lignite and bituminous coals from nonendemic regions in Romania and the USA. Room temperature, hot water bath, and Soxhlet aqueous extraction experiments were conducted between 25 and 80 °C, and from 5 to 128 days in duration. A greater number of organic compounds and in higher concentrations were present in all three types of leaching experiments involving endemic area Pliocene lignite samples compared to all other coals examined. A BEN causing molecule or molecules may be among phenols, PAHs, benzenes, and/or lignin degradation compounds. The proposed transport pathway of the Pliocene lignite hypothesis for organic compound exposure from endemic area Pliocene lignite coals to well and spring drinking water, is likely. Aromatic compounds leached by groundwater from Pliocene lignite deposits in the vicinity of endemic BEN areas may play a role in the etiology of the disease. A better understanding of organic compounds leached by groundwater from Pliocene lignite deposits may potentially lead to the identification and implementation of effective strategies for the prevention of exposure to the causative agent(s) for BEN, and in turn, prevention of the disease.
BEN; Coal geochemistry; Environmental etiology; GC/MS; Groundwater simulation; Leaching experiments; Low-rank coal; Medical geology; Pliocene lignite hypothesis
Balkan endemic nephropathy (BEN) has attracted increasing attention as a possible environmental disease, and a significant amount of research from complementary scientific fields has been dedicated to its etiology. There are two actual competing theories attempting to explain the cause of this kidney disease: 1) the mycotoxin hypothesis, which considers that BEN is produced by ochratoxin A ingested intermittently in small amounts by the individuals in the endemic regions, and 2) the Pliocene lignite hypothesis, which proposes that the disease is caused by long-term exposure to polycyclic aromatic hydrocarbons and other toxic organic compounds leaching into the well drinking water from low rank coals underlying or proximal to the endemic settlements. We outline the current developments and future prospects in the study of BEN and differentiate possible factors and cofactors in disease etiology.
Balkan endemic nephropathy (BEN) represents a chronic progressive interstitial nephritis in striking correlation with uroepithelial tumours of the upper urinary tract. The disease has endemic distribution in the Danube river regions in several Balkan countries.
DNA methylation is a primary epigenetic modification that is involved in major processes such as cancer, genomic imprinting, gene silencing, etc. The significance of CpG island methylation status in normal development, cell differentiation and gene expression is widely recognized, although still stays poorly understood.
We performed whole genome DNA methylation array analysis on DNA pool samples from peripheral blood from 159 affected individuals and 170 healthy individuals. This technique allowed us to determine the methylation status of 27 627 CpG islands throughout the whole genome in healthy controls and BEN patients. Thus we obtained the methylation profile of BEN patients from Bulgarian and Serbian endemic regions.
Using specifically developed software we compared the methylation profiles of BEN patients and corresponding controls and revealed the differently methylated regions. We then compared the DMRs between all patient-control pairs to determine common changes in the epigenetic profiles.
SEC61G, IL17RA, HDAC11 proved to be differently methylated throughout all patient-control pairs. The CpG islands of all 3 genes were hypomethylated compared to controls. This suggests that dysregulation of these genes involved in immunological response could be a common mechanism in BEN pathogenesis in both endemic regions and in both genders.
Our data propose a new hypothesis that immunologic dysregulation has a place in BEN etiopathogenesis.
Epigenetics; Whole genome array analysis; Balkan endemic nephropathy
Aristolochic acids (AAs) are a structurally-related family of nephrotoxic and carcinogenic nitrophenanthrene compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs have been implicated in the etiology of so-called Chinese herbs nephropathy and of Balkan endemic nephropathy. Both of these disease syndromes are associated with carcinomas of the upper urinary tract (UUC). 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I) is a principal component of Aristolochia herbs. Following metabolic activation, AA-I reacts with DNA to form aristolactam (AL-I)-DNA adducts. We have developed a sensitive analytical method, using ultra-performance liquid chromatography-electrospray ionization/multistage mass spectrometry (UPLC-ESI/MSn) with a linear quadrupole ion-trap mass spectrometer, to measure 7-(deoxyadenosin-N6-yl) aristolactam I (dA-AL-I) and 7-(deoxyguanosin-N2-yl) aristolactam I (dG-AL-I) adducts. Using 10 μg of DNA for measurements, the lower limits of quantitation of dA-AL-I and dG-AL-I are, respectively, 0.3 and 1.0 adducts per 108 DNA bases. We have used UPLC-ESI/MSn to quantify AL-DNA adducts in tissues of rodents exposed to AA, and in the renal cortex of patients with UUC who reside in Taiwan, where the incidence of this uncommon cancer is the highest reported for any country in the world. In human tissues, dA-AL-I was detected at levels ranging from 9 to 338 adducts per 108 DNA bases, whereas dG-AL-I was not found. We conclude that UPLC-ESI/MSn is a highly sensitive, specific and robust analytical method, positioned to supplant 32P-postlabeling techniques currently used for biomonitoring of DNA adducts in human tissues. Importantly, UPLC-ESI/MSn could be used to document exposure to AA, the toxicant responsible for AA nephropathy and its associated UUC.
Although human bitter taste perception is hypothesized to be a dietary adaptation, little is known about genetic signatures of selection and patterns of bitter taste perception variability in ethnically diverse populations with different diets, particularly from Africa. To better understand the genetic basis and evolutionary history of bitter taste sensitivity, we sequenced a 2,975 bp region encompassing TAS2R38, a bitter taste receptor gene, in 611 Africans from 57 populations in West Central and East Africa with diverse subsistence patterns, as well as in a comparative sample of 132 non-Africans. We also examined the association between genetic variability at this locus and threshold levels of phenylthiocarbamide (PTC) bitterness in 463 Africans from the above populations to determine how variation influences bitter taste perception. Here, we report striking patterns of variation at TAS2R38, including a significant excess of novel rare nonsynonymous polymorphisms that recently arose only in Africa, high frequencies of haplotypes in Africa associated with intermediate bitter taste sensitivity, a remarkably similar frequency of common haplotypes across genetically and culturally distinct Africans, and an ancient coalescence time of common variation in global populations. Additionally, several of the rare nonsynonymous substitutions significantly modified levels of PTC bitter taste sensitivity in diverse Africans. While ancient balancing selection likely maintained common haplotype variation across global populations, we suggest that recent selection pressures may have also resulted in the unusually high level of rare nonsynonymous variants in Africa, implying a complex model of selection at the TAS2R38 locus in African populations. Furthermore, the distribution of common haplotypes in Africa is not correlated with diet, raising the possibility that common variation may be under selection due to their role in nondietary biological processes. In addition, our data indicate that novel rare mutations contribute to the phenotypic variance of PTC sensitivity, illustrating the influence of rare variation on a common trait, as well as the relatively recent evolution of functionally diverse alleles at this locus.
adaptive evolution; genotype–phenotype association; African genetic diversity; excess of rare mutations
Benzimidazole anti-microtubule drugs, such as benomyl, induce paralysis and slow the growth of the nematode Caenorhabditis elegans. We have identified 28 mutations in C. elegans that confer resistance to benzimidazoles. All resistant mutations map to a single locus, ben-1. Virtually all these mutations are genetically dominant. Molecular cloning and DNA sequence analysis established that ben-1 encodes a beta- tubulin. Some resistant mutants are completely deleted for the ben-1 gene. Since the deletion strains appear to be fully resistant to the drugs, the ben-1 product appears to be the only benzimidazole-sensitive beta-tubulin in C. elegans. Furthermore, since animals lacking ben-1 are viable and coordinated, the ben-1 beta-tubulin appears to be nonessential for growth and movement. The ben-1 function is likely to be redundant in the nematode genome.
The 25 human bitter receptors and their respective genes (TAS2Rs) contain unusually high levels of allelic variation, which may influence response to bitter compounds in the food supply. Phenotypes based on the perceived bitterness of single bitter compounds were first linked to food preference over 50 years ago. The most studied phenotype is propylthiouracil bitterness, which is mediated primarily by the TAS2R38 gene and possibly others. In a laboratory-based study, we tested for associations between TAS2R variants and sensations, liking, or intake of bitter beverages among healthy adults who were primarily of European ancestry. A haploblock across TAS2R3, TAS2R4, and TAS2R5 explained some variability in the bitterness of espresso coffee. For grapefruit juice, variation at a TAS2R19 single nucleotide polymorphism (SNP) was associated with increased bitterness and decreased liking. An association between a TAS2R16 SNP and alcohol intake was identified, and the putative TAS2R38–alcohol relationship was confirmed, although these polymorphisms did not explain sensory or hedonic responses to sampled scotch whisky. In summary, TAS2R polymorphisms appear to influence the sensations, liking, or intake of common and nutritionally significant beverages. Studying perceptual and behavioral differences in vivo using real foods and beverages may potentially identify polymorphisms related to dietary behavior even in the absence of known ligands.
alcohol drinking; alleles; bitter taste receptors; food choice; genetics
Taste and oral sensations vary in humans. Some of this variation has a genetic basis, and two commonly measured phenotypes are the bitterness of propylthiouracil (PROP) and the number of fungiform papillae on the anterior tongue. While the genetic control of fungiform papilla is unclear, PROP bitterness associates with allelic variation in the taste receptor gene, TAS2R38. The two common alleles are AVI and PAV (proline, alanine, valine, and isoleucine); AVI/AVI homozygotes taste PROP as less bitter than heterozygous or homozygous PAV carriers. In this laboratory-based study, we determined whether taste of a bitter probe (quinine) and vegetable intake varied by taste phenotypes and TAS2R38 genotype in healthy adults (mean age=26 years). Vegetable intake was assessed via two validated, complementary methods: food records (Food Pyramid servings standardized to energy intake) and food frequency questionnaire (general intake question and composite vegetable groups). Quinine bitterness varied with phenotypes but not TAS2R38; quinine was more bitter to those who tasted PROP as more bitter or had more papillae. Nontasters by phenotype or genotype reported greater consumption of vegetables, regardless of type (i.e., the effect generalized to all vegetables and was not restricted to those typically thought of as being bitter). Furthermore, nontasters with more papillae reported greater vegetable consumption than nontasters with fewer papillae, suggesting that when bitterness does not predominate, more papillae enhance vegetable liking. These findings suggest that genetic variation in taste, measured by multiple phenotypes or TAS2R38 genotype, can explain differences in overall consumption of vegetables, and this was not restricted to vegetables that are predominantly bitter.
Taste; Genetics; Food preferences; Vegetables; Propylthiouracil; Fungiform papillae
Human perception of bitterness displays pronounced interindividual variation. This phenotypic variation is mirrored by equally pronounced genetic variation in the family of bitter taste receptor genes. To better understand the effects of common genetic variations on human bitter taste perception, we conducted a genome-wide association study on a discovery panel of 504 subjects and a validation panel of 104 subjects from the general population of São Paulo in Brazil. Correction for general taste-sensitivity allowed us to identify a SNP in the cluster of bitter taste receptors on chr12 (10.88– 11.24 Mb, build 36.1) significantly associated (best SNP: rs2708377, P = 5.31 × 10−13, r2 = 8.9%, β = −0.12, s.e. = 0.016) with the perceived bitterness of caffeine. This association overlaps with—but is statistically distinct from—the previously identified SNP rs10772420 influencing the perception of quinine bitterness that falls in the same bitter taste cluster. We replicated this association to quinine perception (P = 4.97 × 10−37, r2 = 23.2%, β = 0.25, s.e. = 0.020) and additionally found the effect of this genetic locus to be concentration specific with a strong impact on the perception of low, but no impact on the perception of high concentrations of quinine. Our study, thus, furthers our understanding of the complex genetic architecture of bitter taste perception.
Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In ∼50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA→T and AL-II-dG→T transversions.
Previous reports that the sensitivity to the bitter tasting substance 6-n-propylthiouracil (PROP) is related to the sensitivity to other tastes, to chemical irritants, and to fats and oils have led to adoption of PROP as a measure of general oral sensitivity and as a predictor of dietary habits that could impact health. The results, however, have not been consistent. It was recently discovered that the ability to perceive “thermal taste” (i.e., sweetness from thermal stimulation alone) was associated with higher responsiveness to 4 prototypical taste stimuli but not to PROP. This finding implied that individual differences in taste perception are determined in large part by factors other than those related to genetic expression of the PROP receptor. The present study followed up this observation by comparing individual differences in perception of 4 prototypical taste stimuli (sucrose, NaCl, citric acid, and quinine) and PROP under conditions that also enabled assessment of the reliability of individual intensity ratings of taste. Creaminess ratings of 3 milk products that had different fat contents were also collected to investigate further the relationship between taste and oral somatosensory perception. The results showed that intensity ratings across 2 trials were significantly correlated for all 5 taste stimuli and that averaging across replicates led to significant correlations among the 4 prototypical stimuli. In contrast, the bitterness of PROP was correlated only with the bitterness of quinine. None of the taste stimuli, including PROP, was significantly correlated with ratings of creaminess. These results imply 1) that with the exception of PROP, as few as 2 intensity ratings of common taste stimuli can reveal individual differences in overall taste perception and 2) that any relationship between taste and oral sensation is too weak to be detected under the same conditions. Accordingly, the results support other evidence that the genetic factors which determine the ability to perceive PROP do not play a major role in overall taste and oral somatosensory perception.
central neural process; individual differences; PROP; taste sensitivity; test–retest reliability
Phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP), chemically related compounds, are probes for genetic variation in bitter taste, although PROP is safer with less sulfurous odor. Threshold for PROP distinguishes nontasters (increased threshold) from tasters (lower threshold); perceived intensity subdivides tasters into medium tasters (PROP is bitter) and supertasters (PROP is very bitter). Compared with supertasters, nontasters have fewer taste papillae on the anterior tongue (fungiform papillae) and experience less negative (e.g., bitterness) and more positive (eg, sweetness) sensations from alcohol. We determined whether the TAS2R38 gene at 7q36 predicted PROP bitterness, alcohol sensation and use.
Healthy adults (53 women, 31 men; mean age 36 years)—primarily light and moderate drinkers—reported the bitterness of five PROP concentrations (0.032–3.2 mM) and intensity of 50% ethanol on the general Labeled Magnitude Scale. PROP threshold and density of fungiform papillae were also measured. Subjects had common TAS2R38 gene haplotypes [alanine-valine-isoleucine (AVI) and proline-alanine-valine (PAV)].
PROP bitterness varied significantly across genotypes with repeated measures ANOVA: 26 AVI/AVI homozygotes tasted less bitterness than either 37 PAV/AVI heterozygotes or 21 PAV/PAV homozygotes. The PAV/PAV group exceeded the PAV/AVI group for bitterness only for the top PROP concentrations. The elevated bitterness was musch less than if we defined the groups using psychophysical criteria. With multiple regression analyses, greater bitterness from 3.2 mM PROP was a significant predictor of greater ethanol intensity and less alcohol intake—effects separate from age and sex. Genotype was a significant predictor of alcohol intake, but not ethanol intensity. With ANOVA, AVI/AVI homozygotes reported higher alcohol use than either PAV/AVI heterozygotes or PAV/PAV homozygotes. When age effects were minimized, PROP bitterness explained more variance in alcohol intake than did the TAS2R38 genotype.
These results support taste genetic effects on alcohol intake. PROP bitterness serves as a marker of these effects.
Taste; Alcohol Consumption; Genetics; PTC/PROP Bitterness; TAS2R38
Genetic screens for Drosophila mutants defective in pavlovian olfactory memory have provided unique insight into the molecular basis of memory storage. Occasionally, these singular genetic lesions have been assembled into meaningful molecular pathways and neural circuitries. For the most part, however, these genes and their expression patterns in the CNS remain fragmented, demanding new clues from continued mutant screens. From a behavioral screen for long-term memory (LTM) mutants, we have identified ben (CG32594), which encodes a novel protein. Mutations of ben specifically disrupt LTM, leaving earlier memory phases intact. The role of ben appears physiological rather than developmental, because acutely induced expression of a ben+ transgene in adults rescues the mutant’s LTM defect. More interestingly, induced expression of ben+ specifically in mushroom bodies (MBs), but not in the ellipsoid body of the central complex, is sufficient to rescue the mutant LTM defect. This suggests a role for ben in the MB during olfactory memory formation. We also provide evidence that BEN interacts genetically in both synaptic transmission and LTM formation with SCAMP, a synaptic protein known to be involved in vesicle recycling.
olfactory learning; synaptic transmission mutants; larval neuromuscular junction; mutant; learning and memory; neurotransmission