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Biomarkers of cadmium and arsenic interactions
Toxicology and Applied Pharmacology 206 (2005) 191 – 197 www.elsevier.com/locate/ytaap
Biomarkers of cadmium and arsenic interactions G.F. Nordberga,T, T. Jina,b, F. Hongc, A. Zhangc, J.P. Buchetd, A. Bernardd a
Environmental Medicine, Umea˚ University, S-901 87 Umea˚, Sweden Department of Occupational Health, School of Public Health, Fudan University, Shanghai, 200032 Shanghai, PR China c Department of Toxicology, School of Public Health, Guiyang Medical College, Guiyang 550004, PR China d Universite´ Catholique de Louvain, Brussels 1200, Belgium
b
Received 17 March 2004; accepted 4 November 2004 Available online 8 April 2005
Abstract Advances in proteomics have led to the identification of sensitive urinary biomarkers of renal dysfunction that are increasingly used in toxicology and epidemiology. Recent animal data show that combined exposure to inorganic arsenic (As) and cadmium (Cd) gives rise to more pronounced renal toxicity than exposure to each of the agents alone. In order to examine if similar interaction occurs in humans, renal dysfunction was studied in population groups (619 persons in total) residing in two metal contaminated areas in China: mainly a Cd contaminated area in Zhejiang province (Z-area) and mainly a As contaminated area in Guizhou province (G-area). Nearby control areas without excessive metal exposure were also included. Measurements of urinary h2-microglobulin (UB2MG), N-acetyl-h-glucosaminidase (UNAG), retinol binding protein (URBP) and albumin (UALB) were used as markers of renal dysfunction. Urinary Cd (UCd) and total As (UTAs) were analyzed by graphite-furnace atomic absorption spectrometry. Urinary inorganic As and its mono- and di-methylated metabolites (UIAs) were determined by Hydride generation. Results. As expected, the highest UCd values occurred in Z-area (Geometric mean, GM 11.6 Ag/g crea) while the highest UTAs values occurred in G-area (GM = 288 Ag/g crea). Statistically significant increases compared to the respective control area were present both for UTAs, UCd and for UB2MG, UNAG and UALB in Z-area as well as in G-area. UIAs was determined only in Z area. In G-area, there was a clear dose–response pattern both in relation to UTAs and UCd for each of the biomarkers of renal dysfunction. An interaction effect between As and Cd was demonstrated at higher levels of a combined exposure to As and Cd enhancing the effect on the kidney. In Z-area an increased prevalence of B2MG-uria, NAG-uria and ALB-uria was found in relation to UCd, but no relationship to UTAs was found. A statistically significant relationship between UIAs and UB2MG was found among women in this area and an interaction between As and Cd was indicated for B2MG. Conclusion. The present studies, which employed sensitive biomarkers of renal dysfunction, give support to the idea that human coexposure to Cd and inorganic arsenic gives rise to more pronounced renal damage than exposure to each of the elements alone, but further studies are needed to establish and clarify this interaction. D 2005 Elsevier Inc. All rights reserved. Keywords: Human health effects; Toxicity; Cadmium; Arsenic; Urinary proteins; Renal dysfunction
Introduction The toxicology of arsenic and cadmium has been extensively studied both in experimental animals and humans (ATSDR, 2000; Jarup et al., 1998). Arsenic is No 1 and
T Corresponding author. Fax: +46 90 7851705. E-mail address: [email protected] (G.F. Nordberg). 0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.11.028
cadmium No 7 in the Priority list of Hazardous substances in USA (ATSDR, 2001). These metals often occur together as contaminants in soil and water (Fay and Mumtaz, 1996). The critical effect in humans exposed to cadmium occupationally or environmentally is renal dysfunction (Jarup et al., 1998). Only limited evidence of renal toxicity of arsenic (kidney cancer excepted) is available from animal models using relatively high doses. However, studies in mice have reported that chronic exposure to cadmium produces more
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China. The present report is based on data from two previous studies (Buchet et al., 2003; Hong et al., 2004) in which sensitive biomarkers of renal dysfunction were employed. These biomarkers had been developed based on achievements in applied proteomics (Bernard, 2004; Bernard et al., 1992, 1994; Jin et al., 1999) and included urinary albumin as a biomarker for glomerular damage and the urinary level of the following proteins as biomarkers of renal tubular dysfunction: urinary retinol binding protein (RBP), beta-2-microglobulin (B2M), and N-acetyl-h-dglucosaminidase (NAG).
Materials and methods
Fig. 1. Areas polluted by Arsenic and Cadmium in China.
renal toxicity than arsenic and the combination of cadmium and arsenic produces even more renal injury than either of the chemicals given alone (Liu et al., 2000). In Guizhou province, China, the domestic use of arsenic containing coal gives rise to high arsenic concentrations in ambient air and in some vegetables dried while exposed to the coal smoke, for example, 1000 mg/kg in capsicum. Arsenic in drinking water is low, less than 50 Ag/L (Zhang et al., 2000). Consumption of fish and shrimp is very low in these areas, and therefore the dominate intake of arsenic is in the inorganic form from other kinds of food. Human health protection most frequently employs exposure limits based on criteria for single agents (WHO, 2002). Exposures, however, often are to mixtures of organic and inorganic compounds and interactions among chemicals may be important. Nevertheless, information on the toxicity of the mixture is often lacking. Exposures to inorganic substances usually involve multiple compounds (Snow, 1992). For instance, arsenic (As) and cadmium (Cd) co-exposure frequently occurs in a variety of settings (Diaz-Barriga et al., 1993; IARC, 1993). As and Cd are also by-products obtained from processing other metals, leading to common co-exposure in industrial settings (IARC, 1993). Thus, there is a clear potential for simultaneous or sequential exposure to As and Cd in general populations and at workplaces. Interactions between As and Cd have been reported that result in acute liver injury (Hochadel and Waalkes, 1997) and in renal injury in mice (Liu et al., 2000), but little is known about their potential interaction in renal dysfunction in human populations, especially from chronic exposure. The primary goal of the present studies was to characterize the interaction of As and Cd in producing renal dysfunction in humans exposed to combinations of these two agents in
Study population. In China, a considerable number of areas contaminated by As or Cd have been identified (Fig. 1). In some areas, there are combined contamination and subsequent human exposure to As and Cd. The present study was performed in a mainly As contaminated area with some Cd exposure in Guizhou province, China (Garea) and a mainly Cd polluted area with some As exposure in Zhejiang province, China (Z-area). For each of the areas, age- and sex-matched control groups in the same province, not contaminated by metals, were also studied in order to allow comparison between exposed and non-exposed (i.e., control groups) and to provide low exposure observations for dose–response analyses. By a three-dimensional display of the data, possible interactions between As and Cd exposures in inducing renal dysfunction were examined. In G-area where burning coal contains As, concentrations of As is about 100–8300 mg/kg in coal, 16 mg/kg in soil. The average arsenic content of ambient air has been reported as 0.22 mg/m3 and there is 1.8 mg/kg in rice, 11 mg/kg in corn and 1096 mg/kg in capsicum (Hong et al., 2004). Cadmium concentrations were 0.02, 0.06, 0.11, 0.17 and 2.1 mg/kg in corn, capsicum, soil, rice and coal, respectively. And arsenic and cadmium levels in the tobacco were 0.39 mg/kg and 2.00 mg/kg, respectively. A nearby control area without excessive metal exposure was also included (As in
Table 1 Geometric means of urinary cadmium (UCd) and urinary arsenic (UAs) by gender for the exposed group and the control area in Guizhou province, China (G-area) UCd and As in residents living in G area
Control
Exposed group
T P b 0.01.
Sex
N
UAs (Ag/g Cr)
UCd (Ag/g Cr)
Males Females Total Males Females Total
68 55 123 72 50 122
60.67 51.76 56.23 278.61T 325.84T 288.40T
0.79 0.94 0.86 2.22T 2.06T 2.16T
G.F. Nordberg et al. / Toxicology and Applied Pharmacology 206 (2005) 191–197 Table 2 Geometric means of Urinary proteins by gender from the Control area (Control) and the polluted area (Exposed group) in Guizhou province (Garea) China Renal function in G area
Control
Exposed group
Sex
N
Uh2-MG (Ag/gCr)
UNAG (U/gCr)
UALB (mg/gCr)
M F T M F T
68 55 123 72 50 122
125.03 106.66 114.81 220.80T 196.79T 213.80T
4.30 3.17 3.68 12.37T 10.45T 11.88T
4.89 4.05 4.49 13.49T 12.71T 13.12T
Note. Uh2-MG: urinary beta-2-microglobulin; UNAG: urinary N-acetyl-hd-glucosaminidase; UALB: urinary albumin. T P b 0.01.
capsicum 0.5 mg/kg and in rice 0.4 mg/kg, Cd in capsicum 0.03 and in rice 0.04 mg/kg). Participants were randomly selected from people living since birth in the contaminated and control areas matched for age and sex. The total number of participants in G area was 245, made up of 122 in the polluted area (72 men and 50 women) and 123 in the control area (68 men and 55 women) cf. Table 1. Z-area is a mainly Cd contaminated area with some As exposure. It is located in Zhejiang province. A nearby control area was also studied. Cd levels in rice were 2.4 mg/ kg in the contaminated area and 0.05 mg/kg in the control area (Jin et al., 2002; Nordberg et al., 2002). Levels of As in rice were 0.14 mg/kg in the contaminated area and 0.09 mg/ kg in the control area. Persons who had resided in the respective areas since birth were selected as described previously (Jin et al., 2002). The present study included 195 persons (91 men and 104 women) from the heavily Cd polluted area and 179 persons (79 men and 100 women) from the control area. The numbers are slightly fewer than in the original study (Jin et al., 2002; Nordberg et al., 2002) because of the lack of urine samples for As analysis. Only
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persons with data on both Cd and As in urine were included in the present study. Subjects were asked to answer a detailed questionnaire by trained and supervised interviewers. No statistically significant differences were observed between exposed vs. controls in the respective study areas in the social and economic status. Subjects were asked to answer a detailed questionnaire by trained and supervised interviewers. The study was carried out with permission of the local authority and the ethics committee of Shanghai Medical University or Fudan University, Shanghai, China (Hong et al., 2004). For the study in Z-area approval was also obtained from the ethics committee of the medical faculty of Umea University, Sweden (Buchet et al., 2003; Jin et al., 2002; Nordberg et al., 2002). Informed consent was obtained from each participating individual. Urine collection and analytical method. Urine samples were collected from all participants and were kept frozen at 20 8C until analysis. Each urine sample was divided into three parts immediately after collection. Of these, a part, which was used for arsenic and cadmium measurement, was acidified with concentrated nitric acid. A part was treated with 0.1 M NaOH and was used for the measurement of h2MG. The third part was assayed for ALB, NAG and creatinine without pre-treatment. UAs and UCd concentrations were measured by graphite-furnace atomic absorption spectrometry (AAS, David et al., 1991; Jin et al., 2002). For analytical quality assurance, both calibration standards and one run of reference materials (Seronorm Trace Elements Urine, Oslo. Norway) were used for every set of analyses performed. Urinary Cd was determined using the standard addition method with proven accuracy (Jin et al., 2002). Urinary Inorganic Arsenic (UIAs) and its metabolites were determined by hydride generation and atomic fluorescence spectrometry and URBP by latex immunoassay (Buchet et al., 2003). Uh2MG and UALB
Fig. 2. Prevalence (%) of elevated urinary excretion (above cut off level) of biomarker proteins by Urinary Cadmium (UCd) and by Urinary Arsenic in G-area. Footnote: Uh2MG: Urinary beta-2-microglobulin, UNAG: Urinary N-acetylglucosaminidase, UALB: Urinary albumin.
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Fig. 3. Prevalence (%) of elevated excretion for three biomarker proteins in G-area in relation to combined exposure to Cd and As as indicated by urinary As and Cd excretion. Footnote: UB2MG: Urinary beta-2-microglobulin, UNAG: Urinary n-acetylglucosaminidase, UALB: Urinary albumin.
and UMT (urinary metallothionein) were measured by ELISA (Neuman and Cohen, 1989) and UNAG was measured as described by Price (1992). h2MG, ALB and NAG kits were purchased from the Debo Bioengineering Ltd., China. Creatinine was measured by the Jaffe reaction method (Hare, 1950). All urinary parameters were adjusted for creatinine in urine. Cut-off points for renal dysfunction biomarkers (proteinuria). B2M-uria, RBP-uria, NAG-uria and ALBuria were defined as urinary excretion of the respective proteins above the cut-off point. The cut-off point was defined based on the upper 5% limit value in the nonpolluted area. The cut-off values of Uh2MG, URBP, UNAG and UALB were 0.30 mg/g creatinine, 0.3 mg/g creatinine, 23.00 U/g creatinine and 15.00 mg/g creatinine, respectively. Statistical analysis. Procedures of the SPSS version 11.0 software were used for frequency means, correlation, variance, regression analyses. The cut-off points (abnormal values) for the criterion variables were defined as the 95% upper limit values, which were calculated from the control group. For comparisons between more than two groups, a one-way analysis of variance (ANOVA) was used. Distributions of the biological measurements were normalized by logarithmic transformation. Data were expressed in terms of geometric means.
Results The concentrations of arsenic and cadmium in urine by gender in G area are shown in Table 1. UAs and UCd concentrations in the polluted areas were significantly higher than those in the control areas. In the polluted area, the geometric mean of UAs and UCd were 288 Ag/g creatinine and 2.16 Ag/g creatinine, respectively. The h2MG, ALB and NAG levels in urine are shown in Table 2. The levels of Uh2MG, UALB and UNAG in the
polluted area were significantly higher than those in the control area. There were no significant differences in UAs, UCd and urinary parameters of renal dysfunction between males and females within the exposed or within the control area. In Fig. 2, the prevalence of B2M-uria, NAG-uria and ALB-uria is shown in various UAs strata (0, 50, 100, 200, 400 Ag/g creatinine) and UCd strata (0, 1.0, 2.00, 5.00 Ag/g creatinine) in G area. A clear dose–response pattern is evident both in relation to UAs and UCd for each of the biomarkers of renal dysfunction. In Fig. 3, the combined effect of Cd and As is illustrated. There were no subjects exposed to low UAs. An interaction effect between As and Cd is illustrated, at higher levels (200 Ag/g creatinine) of a combined exposure to As and Cd demonstrating an enhancing effect of Cd on As-induced renal dysfunction. UCd and UAs levels in the Z area are shown in Table 3. Higher values of As as well as for Cd are found in the exposed group compared to the control group. Results of determinations of urinary protein biomarkers in participants from G-area are given in Table 4. For each of UB2M,
Table 3 Urinary levels of Cadmium (UCd) and Arsenic (As) in residents living in the Control area in Zhejiang province (Z-area) and in residents in the heavily polluted area (Exposed group) UCd and As in residents living in Z area
Control
Exposed group
Sex
N
UCd (Ag/gCr)
UAs (Ag/gCr)
M F T M F T
79 100 179 91 104 195
2.17 1.86 1.99 10.08T 13.11T, TT 11.61T
61.96 53.16 56.87 132.07T 176.20T, TT 154.03T
M = male participants, F = female participants T = total number of participants. Open squares indicate statistical significance in relation to control group. Filled triangle indicates statistical significance between males and females. T P b 0.01. TT P b 0.05.
G.F. Nordberg et al. / Toxicology and Applied Pharmacology 206 (2005) 191–197 Table 4 Results from Z-area: urinary excretion of proteins used as biomarkers of renal dysfunction in persons residing in the polluted area (Exposed Group) and in the nearby control area (control) Kidney function in residents living in Z area
Control
Exposed group
Sex
N
Uh2-MG (Ag/gCr)
UNAG (U/gCr)
UALB (mg/gCr)
M F T M F T
79 100 179 91 104 195
209.48 180.59 193.16 718.70T 651.58T 682.67T
2.96 2.72 2.82 7.25T 10.77T, TTT 8.97T
3.28 3.61 3.46 5.70TT 9.21T, TTT 7.39T
Open squares indicate statistical significance in relation to controls. Filled triangles indicate statistical significance in relation to males. Uh2-MG: Urinary beta-2-microglobulin; UNAG: Urinary N-acetyl-h-dglucosaminidase; UALB: Urinary albumin. T P b 0.01. TT P b 0.05. TTT P b 0.01.
UNAG and UALB, there were statistically significant higher values in the exposed group compared to controls (Table 4). Women had higher levels than men of UNAG and UALB in the exposed area (Table 4). The prevalence of elevated excretion of the respective proteins (above the cut off point) were plotted in relation to UCd and As and shown in (Fig. 4) in Z area. In general, a dose–response pattern is seen. The dose–response pattern is somewhat more consistent for UCd. When plotting the prevalence of each biomarker in relation to combined exposure to UAs and UCd (Fig. 5), a dose–response relationship in relation to UCd remains, but there is no relationship with (total) UAs (Fig. 5). Since Z area is a coastal area and consumption of fish and seafood is prevalent, it is likely that UAs to a large extent represents excretion of organic arsenic compounds occurring in fish and other seafood. Such arsenic compounds are virtually non-toxic and their occurrence in urine
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masks a possible effect of inorganic As. In order to examine the specific effect of Inorganic arsenic for Z area, UIAs was determined by hydride generation on the same urine samples. Urinary excretion of the renal biomarkers RBP, B2M and NAG were examined in relation to UIAs by multiple regression analyses (Buchet et al., 2003). A statistically significant relationship was found between all three urinary biomarkers and UCd among men and women (data not shown here). A statistically significant relationship between UIAs and URBP or UB2M was found among women (but not among men) and there was also evidence for an interaction between IAs and Cd among women (Table 5, Buchet et al., 2003).
Discussion The present study in two metal (and metalloid) contaminated areas in China provides evidence that renal dysfunction was induced in humans in a dose-related pattern by the exposures occurring in these areas. The main contribution of the present studies is in relation to a mutually enhancing interaction between As and Cd exposures in inducing renal dysfunction in humans. Although there was a lack of persons with low As exposure and high Cd exposure in Garea, a clear enhancing effect of simultaneous Cd exposure was demonstrated in subgroups with high UAs. There was a lack of persons with low arsenic exposure but at higher As exposures a clear enhancing effect of simultaneous Cd exposure was demonstrated. Also in Z-area, an interaction between Cd and inorganic As was indicated. The reason that interaction was statistically significant only among women is probably related to the fact that renal tubular toxicity occurred with higher prevalence among women than men, the number of men studied was not sufficiently large to achieve statistical significance. The data for both areas thus
Fig. 4. Prevalence of elevated urinary excretion of biomarker proteins in Z-area. Footnote: UB2-MG: Urinary beta-2-microglobulin; UNAG: Urinary N-acetylh-d-glucosaminidase; UALB: Urinary albumin.
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Fig. 5. Prevalence of elevated urinary excretion of biomarker proteins in Z area. Footnote: UB2-MG: Urinary beta-2-microglobulin; UNAG: Urinary N-acetylh-d-glucosaminidase; UALB: Urinary albumin.
indicate that combined exposure to inorganic As and Cd gives rise to a higher prevalence of renal dysfunction than either of the exposures alone. The potentiation by arsenic of cadmium nephrotoxicity previously observed in mice included a statistically significant additivity and/or synergism between the two elements (Liu et al., 2000) and a similar situation is observed in humans in our study. The interactions illustrated by the evidence presented in the present paper are further supported by data recently reported by Hong et al. (2004) for G-area and by Jin et al. (2004) for Z-area. The benchmark dose of Cd required for renal dysfunction to occur was much lower in the heavily As contaminated G-area (Hong et al., 2004) than in the mainly Cd contaminated Z-area (Jin et al., 2004). In addition to the observations concerning interactions, it is interesting that in area G, where arsenic exposure was most prominent, effects on albumin excretion, a biomarker of glomerular dysfunction was most prevalent. It has not previously been clearly demonstrated in humans that environmental exposures to arsenic give rise to renal glomerular dysfunction. It is most probable that Asexposures in G-area are predominantly inorganic As. However, the determinations of UAs were only for total Table 5 Relationship between UCd, UIAs and RBP, B2M among women in Z-area (Data from Buchet et al., 2003) UCd, UIAs and RBP and B2M in women living in Z area
UCd b 4.8 Ag/gCr UCd N 4.8 Ag/gCr a
UIAs (Ag/gCr).
As As As As
b N b N
36a 36a 36a 36a
N
RBP (mg/gCr)
B2M (mg/gCr)
124 87 75 130
0.059 0.083 0.077 0.163
0.140 0.189 0.202 0.344
As. Since G-area is an inland area where consumption of fish and shellfish is very low, and exposure to inorganic As is high, it is still reasonable to assume that UAs values reflect mainly inorganic As. Renal tubular toxicity has been well documented previously in humans exposed for a long time to environmental Cd (Jarup et al., 1998; Jin et al., 2004; Nordberg, 2004; Nordberg et al., 2002). In the present study, renal tubular dysfunction, indicated by a high prevalence of elevated excretion of NAG and B2M, occurred in both Gand Z-areas among persons with high exposure to Cd and low exposure to As. It is interesting to note that also among persons with predominant As-exposure (and low exposure to Cd), there was increased prevalence of renal tubular dysfunction biomarkers (increased UB2M and UNAG). Such a relationship between As exposure and renal tubular dysfunction in humans has not been well documented previously. There is a need to confirm the presently reported findings by additional epidemiological studies employing improved methods for determination of As in urine and possibly improved biomarkers of biological damage and dysfunction on a larger sample of the exposed populations.
Conclusions ! The present studies, which employed sensitive biomarkers of renal dysfunction, give support to the idea that human co-exposure to Cd and inorganic As gives rise to more pronounced renal damage than exposure to each element alone. ! Further studies, possibly including additional new biomarkers and improved exposure assessment technology, are needed to firmly establish this interaction.
G.F. Nordberg et al. / Toxicology and Applied Pharmacology 206 (2005) 191–197
Acknowledgments This study was funded by the European Commission INCO-DC programme (No.ERB3514PL971430) and by the National Natural Science Foundation of China (No.30060077/C030108).
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Biomarkers of cadmium and arsenic interactions G.F. Nordberga,T, T. Jina,b, F. Hongc, A. Zhangc, J.P. Buchetd, A. Bernardd a
Environmental Medicine, Umea˚ University, S-901 87 Umea˚, Sweden Department of Occupational Health, School of Public Health, Fudan University, Shanghai, 200032 Shanghai, PR China c Department of Toxicology, School of Public Health, Guiyang Medical College, Guiyang 550004, PR China d Universite´ Catholique de Louvain, Brussels 1200, Belgium
b
Received 17 March 2004; accepted 4 November 2004 Available online 8 April 2005
Abstract Advances in proteomics have led to the identification of sensitive urinary biomarkers of renal dysfunction that are increasingly used in toxicology and epidemiology. Recent animal data show that combined exposure to inorganic arsenic (As) and cadmium (Cd) gives rise to more pronounced renal toxicity than exposure to each of the agents alone. In order to examine if similar interaction occurs in humans, renal dysfunction was studied in population groups (619 persons in total) residing in two metal contaminated areas in China: mainly a Cd contaminated area in Zhejiang province (Z-area) and mainly a As contaminated area in Guizhou province (G-area). Nearby control areas without excessive metal exposure were also included. Measurements of urinary h2-microglobulin (UB2MG), N-acetyl-h-glucosaminidase (UNAG), retinol binding protein (URBP) and albumin (UALB) were used as markers of renal dysfunction. Urinary Cd (UCd) and total As (UTAs) were analyzed by graphite-furnace atomic absorption spectrometry. Urinary inorganic As and its mono- and di-methylated metabolites (UIAs) were determined by Hydride generation. Results. As expected, the highest UCd values occurred in Z-area (Geometric mean, GM 11.6 Ag/g crea) while the highest UTAs values occurred in G-area (GM = 288 Ag/g crea). Statistically significant increases compared to the respective control area were present both for UTAs, UCd and for UB2MG, UNAG and UALB in Z-area as well as in G-area. UIAs was determined only in Z area. In G-area, there was a clear dose–response pattern both in relation to UTAs and UCd for each of the biomarkers of renal dysfunction. An interaction effect between As and Cd was demonstrated at higher levels of a combined exposure to As and Cd enhancing the effect on the kidney. In Z-area an increased prevalence of B2MG-uria, NAG-uria and ALB-uria was found in relation to UCd, but no relationship to UTAs was found. A statistically significant relationship between UIAs and UB2MG was found among women in this area and an interaction between As and Cd was indicated for B2MG. Conclusion. The present studies, which employed sensitive biomarkers of renal dysfunction, give support to the idea that human coexposure to Cd and inorganic arsenic gives rise to more pronounced renal damage than exposure to each of the elements alone, but further studies are needed to establish and clarify this interaction. D 2005 Elsevier Inc. All rights reserved. Keywords: Human health effects; Toxicity; Cadmium; Arsenic; Urinary proteins; Renal dysfunction
Introduction The toxicology of arsenic and cadmium has been extensively studied both in experimental animals and humans (ATSDR, 2000; Jarup et al., 1998). Arsenic is No 1 and
T Corresponding author. Fax: +46 90 7851705. E-mail address: [email protected] (G.F. Nordberg). 0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.11.028
cadmium No 7 in the Priority list of Hazardous substances in USA (ATSDR, 2001). These metals often occur together as contaminants in soil and water (Fay and Mumtaz, 1996). The critical effect in humans exposed to cadmium occupationally or environmentally is renal dysfunction (Jarup et al., 1998). Only limited evidence of renal toxicity of arsenic (kidney cancer excepted) is available from animal models using relatively high doses. However, studies in mice have reported that chronic exposure to cadmium produces more
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China. The present report is based on data from two previous studies (Buchet et al., 2003; Hong et al., 2004) in which sensitive biomarkers of renal dysfunction were employed. These biomarkers had been developed based on achievements in applied proteomics (Bernard, 2004; Bernard et al., 1992, 1994; Jin et al., 1999) and included urinary albumin as a biomarker for glomerular damage and the urinary level of the following proteins as biomarkers of renal tubular dysfunction: urinary retinol binding protein (RBP), beta-2-microglobulin (B2M), and N-acetyl-h-dglucosaminidase (NAG).
Materials and methods
Fig. 1. Areas polluted by Arsenic and Cadmium in China.
renal toxicity than arsenic and the combination of cadmium and arsenic produces even more renal injury than either of the chemicals given alone (Liu et al., 2000). In Guizhou province, China, the domestic use of arsenic containing coal gives rise to high arsenic concentrations in ambient air and in some vegetables dried while exposed to the coal smoke, for example, 1000 mg/kg in capsicum. Arsenic in drinking water is low, less than 50 Ag/L (Zhang et al., 2000). Consumption of fish and shrimp is very low in these areas, and therefore the dominate intake of arsenic is in the inorganic form from other kinds of food. Human health protection most frequently employs exposure limits based on criteria for single agents (WHO, 2002). Exposures, however, often are to mixtures of organic and inorganic compounds and interactions among chemicals may be important. Nevertheless, information on the toxicity of the mixture is often lacking. Exposures to inorganic substances usually involve multiple compounds (Snow, 1992). For instance, arsenic (As) and cadmium (Cd) co-exposure frequently occurs in a variety of settings (Diaz-Barriga et al., 1993; IARC, 1993). As and Cd are also by-products obtained from processing other metals, leading to common co-exposure in industrial settings (IARC, 1993). Thus, there is a clear potential for simultaneous or sequential exposure to As and Cd in general populations and at workplaces. Interactions between As and Cd have been reported that result in acute liver injury (Hochadel and Waalkes, 1997) and in renal injury in mice (Liu et al., 2000), but little is known about their potential interaction in renal dysfunction in human populations, especially from chronic exposure. The primary goal of the present studies was to characterize the interaction of As and Cd in producing renal dysfunction in humans exposed to combinations of these two agents in
Study population. In China, a considerable number of areas contaminated by As or Cd have been identified (Fig. 1). In some areas, there are combined contamination and subsequent human exposure to As and Cd. The present study was performed in a mainly As contaminated area with some Cd exposure in Guizhou province, China (Garea) and a mainly Cd polluted area with some As exposure in Zhejiang province, China (Z-area). For each of the areas, age- and sex-matched control groups in the same province, not contaminated by metals, were also studied in order to allow comparison between exposed and non-exposed (i.e., control groups) and to provide low exposure observations for dose–response analyses. By a three-dimensional display of the data, possible interactions between As and Cd exposures in inducing renal dysfunction were examined. In G-area where burning coal contains As, concentrations of As is about 100–8300 mg/kg in coal, 16 mg/kg in soil. The average arsenic content of ambient air has been reported as 0.22 mg/m3 and there is 1.8 mg/kg in rice, 11 mg/kg in corn and 1096 mg/kg in capsicum (Hong et al., 2004). Cadmium concentrations were 0.02, 0.06, 0.11, 0.17 and 2.1 mg/kg in corn, capsicum, soil, rice and coal, respectively. And arsenic and cadmium levels in the tobacco were 0.39 mg/kg and 2.00 mg/kg, respectively. A nearby control area without excessive metal exposure was also included (As in
Table 1 Geometric means of urinary cadmium (UCd) and urinary arsenic (UAs) by gender for the exposed group and the control area in Guizhou province, China (G-area) UCd and As in residents living in G area
Control
Exposed group
T P b 0.01.
Sex
N
UAs (Ag/g Cr)
UCd (Ag/g Cr)
Males Females Total Males Females Total
68 55 123 72 50 122
60.67 51.76 56.23 278.61T 325.84T 288.40T
0.79 0.94 0.86 2.22T 2.06T 2.16T
G.F. Nordberg et al. / Toxicology and Applied Pharmacology 206 (2005) 191–197 Table 2 Geometric means of Urinary proteins by gender from the Control area (Control) and the polluted area (Exposed group) in Guizhou province (Garea) China Renal function in G area
Control
Exposed group
Sex
N
Uh2-MG (Ag/gCr)
UNAG (U/gCr)
UALB (mg/gCr)
M F T M F T
68 55 123 72 50 122
125.03 106.66 114.81 220.80T 196.79T 213.80T
4.30 3.17 3.68 12.37T 10.45T 11.88T
4.89 4.05 4.49 13.49T 12.71T 13.12T
Note. Uh2-MG: urinary beta-2-microglobulin; UNAG: urinary N-acetyl-hd-glucosaminidase; UALB: urinary albumin. T P b 0.01.
capsicum 0.5 mg/kg and in rice 0.4 mg/kg, Cd in capsicum 0.03 and in rice 0.04 mg/kg). Participants were randomly selected from people living since birth in the contaminated and control areas matched for age and sex. The total number of participants in G area was 245, made up of 122 in the polluted area (72 men and 50 women) and 123 in the control area (68 men and 55 women) cf. Table 1. Z-area is a mainly Cd contaminated area with some As exposure. It is located in Zhejiang province. A nearby control area was also studied. Cd levels in rice were 2.4 mg/ kg in the contaminated area and 0.05 mg/kg in the control area (Jin et al., 2002; Nordberg et al., 2002). Levels of As in rice were 0.14 mg/kg in the contaminated area and 0.09 mg/ kg in the control area. Persons who had resided in the respective areas since birth were selected as described previously (Jin et al., 2002). The present study included 195 persons (91 men and 104 women) from the heavily Cd polluted area and 179 persons (79 men and 100 women) from the control area. The numbers are slightly fewer than in the original study (Jin et al., 2002; Nordberg et al., 2002) because of the lack of urine samples for As analysis. Only
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persons with data on both Cd and As in urine were included in the present study. Subjects were asked to answer a detailed questionnaire by trained and supervised interviewers. No statistically significant differences were observed between exposed vs. controls in the respective study areas in the social and economic status. Subjects were asked to answer a detailed questionnaire by trained and supervised interviewers. The study was carried out with permission of the local authority and the ethics committee of Shanghai Medical University or Fudan University, Shanghai, China (Hong et al., 2004). For the study in Z-area approval was also obtained from the ethics committee of the medical faculty of Umea University, Sweden (Buchet et al., 2003; Jin et al., 2002; Nordberg et al., 2002). Informed consent was obtained from each participating individual. Urine collection and analytical method. Urine samples were collected from all participants and were kept frozen at 20 8C until analysis. Each urine sample was divided into three parts immediately after collection. Of these, a part, which was used for arsenic and cadmium measurement, was acidified with concentrated nitric acid. A part was treated with 0.1 M NaOH and was used for the measurement of h2MG. The third part was assayed for ALB, NAG and creatinine without pre-treatment. UAs and UCd concentrations were measured by graphite-furnace atomic absorption spectrometry (AAS, David et al., 1991; Jin et al., 2002). For analytical quality assurance, both calibration standards and one run of reference materials (Seronorm Trace Elements Urine, Oslo. Norway) were used for every set of analyses performed. Urinary Cd was determined using the standard addition method with proven accuracy (Jin et al., 2002). Urinary Inorganic Arsenic (UIAs) and its metabolites were determined by hydride generation and atomic fluorescence spectrometry and URBP by latex immunoassay (Buchet et al., 2003). Uh2MG and UALB
Fig. 2. Prevalence (%) of elevated urinary excretion (above cut off level) of biomarker proteins by Urinary Cadmium (UCd) and by Urinary Arsenic in G-area. Footnote: Uh2MG: Urinary beta-2-microglobulin, UNAG: Urinary N-acetylglucosaminidase, UALB: Urinary albumin.
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Fig. 3. Prevalence (%) of elevated excretion for three biomarker proteins in G-area in relation to combined exposure to Cd and As as indicated by urinary As and Cd excretion. Footnote: UB2MG: Urinary beta-2-microglobulin, UNAG: Urinary n-acetylglucosaminidase, UALB: Urinary albumin.
and UMT (urinary metallothionein) were measured by ELISA (Neuman and Cohen, 1989) and UNAG was measured as described by Price (1992). h2MG, ALB and NAG kits were purchased from the Debo Bioengineering Ltd., China. Creatinine was measured by the Jaffe reaction method (Hare, 1950). All urinary parameters were adjusted for creatinine in urine. Cut-off points for renal dysfunction biomarkers (proteinuria). B2M-uria, RBP-uria, NAG-uria and ALBuria were defined as urinary excretion of the respective proteins above the cut-off point. The cut-off point was defined based on the upper 5% limit value in the nonpolluted area. The cut-off values of Uh2MG, URBP, UNAG and UALB were 0.30 mg/g creatinine, 0.3 mg/g creatinine, 23.00 U/g creatinine and 15.00 mg/g creatinine, respectively. Statistical analysis. Procedures of the SPSS version 11.0 software were used for frequency means, correlation, variance, regression analyses. The cut-off points (abnormal values) for the criterion variables were defined as the 95% upper limit values, which were calculated from the control group. For comparisons between more than two groups, a one-way analysis of variance (ANOVA) was used. Distributions of the biological measurements were normalized by logarithmic transformation. Data were expressed in terms of geometric means.
Results The concentrations of arsenic and cadmium in urine by gender in G area are shown in Table 1. UAs and UCd concentrations in the polluted areas were significantly higher than those in the control areas. In the polluted area, the geometric mean of UAs and UCd were 288 Ag/g creatinine and 2.16 Ag/g creatinine, respectively. The h2MG, ALB and NAG levels in urine are shown in Table 2. The levels of Uh2MG, UALB and UNAG in the
polluted area were significantly higher than those in the control area. There were no significant differences in UAs, UCd and urinary parameters of renal dysfunction between males and females within the exposed or within the control area. In Fig. 2, the prevalence of B2M-uria, NAG-uria and ALB-uria is shown in various UAs strata (0, 50, 100, 200, 400 Ag/g creatinine) and UCd strata (0, 1.0, 2.00, 5.00 Ag/g creatinine) in G area. A clear dose–response pattern is evident both in relation to UAs and UCd for each of the biomarkers of renal dysfunction. In Fig. 3, the combined effect of Cd and As is illustrated. There were no subjects exposed to low UAs. An interaction effect between As and Cd is illustrated, at higher levels (200 Ag/g creatinine) of a combined exposure to As and Cd demonstrating an enhancing effect of Cd on As-induced renal dysfunction. UCd and UAs levels in the Z area are shown in Table 3. Higher values of As as well as for Cd are found in the exposed group compared to the control group. Results of determinations of urinary protein biomarkers in participants from G-area are given in Table 4. For each of UB2M,
Table 3 Urinary levels of Cadmium (UCd) and Arsenic (As) in residents living in the Control area in Zhejiang province (Z-area) and in residents in the heavily polluted area (Exposed group) UCd and As in residents living in Z area
Control
Exposed group
Sex
N
UCd (Ag/gCr)
UAs (Ag/gCr)
M F T M F T
79 100 179 91 104 195
2.17 1.86 1.99 10.08T 13.11T, TT 11.61T
61.96 53.16 56.87 132.07T 176.20T, TT 154.03T
M = male participants, F = female participants T = total number of participants. Open squares indicate statistical significance in relation to control group. Filled triangle indicates statistical significance between males and females. T P b 0.01. TT P b 0.05.
G.F. Nordberg et al. / Toxicology and Applied Pharmacology 206 (2005) 191–197 Table 4 Results from Z-area: urinary excretion of proteins used as biomarkers of renal dysfunction in persons residing in the polluted area (Exposed Group) and in the nearby control area (control) Kidney function in residents living in Z area
Control
Exposed group
Sex
N
Uh2-MG (Ag/gCr)
UNAG (U/gCr)
UALB (mg/gCr)
M F T M F T
79 100 179 91 104 195
209.48 180.59 193.16 718.70T 651.58T 682.67T
2.96 2.72 2.82 7.25T 10.77T, TTT 8.97T
3.28 3.61 3.46 5.70TT 9.21T, TTT 7.39T
Open squares indicate statistical significance in relation to controls. Filled triangles indicate statistical significance in relation to males. Uh2-MG: Urinary beta-2-microglobulin; UNAG: Urinary N-acetyl-h-dglucosaminidase; UALB: Urinary albumin. T P b 0.01. TT P b 0.05. TTT P b 0.01.
UNAG and UALB, there were statistically significant higher values in the exposed group compared to controls (Table 4). Women had higher levels than men of UNAG and UALB in the exposed area (Table 4). The prevalence of elevated excretion of the respective proteins (above the cut off point) were plotted in relation to UCd and As and shown in (Fig. 4) in Z area. In general, a dose–response pattern is seen. The dose–response pattern is somewhat more consistent for UCd. When plotting the prevalence of each biomarker in relation to combined exposure to UAs and UCd (Fig. 5), a dose–response relationship in relation to UCd remains, but there is no relationship with (total) UAs (Fig. 5). Since Z area is a coastal area and consumption of fish and seafood is prevalent, it is likely that UAs to a large extent represents excretion of organic arsenic compounds occurring in fish and other seafood. Such arsenic compounds are virtually non-toxic and their occurrence in urine
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masks a possible effect of inorganic As. In order to examine the specific effect of Inorganic arsenic for Z area, UIAs was determined by hydride generation on the same urine samples. Urinary excretion of the renal biomarkers RBP, B2M and NAG were examined in relation to UIAs by multiple regression analyses (Buchet et al., 2003). A statistically significant relationship was found between all three urinary biomarkers and UCd among men and women (data not shown here). A statistically significant relationship between UIAs and URBP or UB2M was found among women (but not among men) and there was also evidence for an interaction between IAs and Cd among women (Table 5, Buchet et al., 2003).
Discussion The present study in two metal (and metalloid) contaminated areas in China provides evidence that renal dysfunction was induced in humans in a dose-related pattern by the exposures occurring in these areas. The main contribution of the present studies is in relation to a mutually enhancing interaction between As and Cd exposures in inducing renal dysfunction in humans. Although there was a lack of persons with low As exposure and high Cd exposure in Garea, a clear enhancing effect of simultaneous Cd exposure was demonstrated in subgroups with high UAs. There was a lack of persons with low arsenic exposure but at higher As exposures a clear enhancing effect of simultaneous Cd exposure was demonstrated. Also in Z-area, an interaction between Cd and inorganic As was indicated. The reason that interaction was statistically significant only among women is probably related to the fact that renal tubular toxicity occurred with higher prevalence among women than men, the number of men studied was not sufficiently large to achieve statistical significance. The data for both areas thus
Fig. 4. Prevalence of elevated urinary excretion of biomarker proteins in Z-area. Footnote: UB2-MG: Urinary beta-2-microglobulin; UNAG: Urinary N-acetylh-d-glucosaminidase; UALB: Urinary albumin.
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Fig. 5. Prevalence of elevated urinary excretion of biomarker proteins in Z area. Footnote: UB2-MG: Urinary beta-2-microglobulin; UNAG: Urinary N-acetylh-d-glucosaminidase; UALB: Urinary albumin.
indicate that combined exposure to inorganic As and Cd gives rise to a higher prevalence of renal dysfunction than either of the exposures alone. The potentiation by arsenic of cadmium nephrotoxicity previously observed in mice included a statistically significant additivity and/or synergism between the two elements (Liu et al., 2000) and a similar situation is observed in humans in our study. The interactions illustrated by the evidence presented in the present paper are further supported by data recently reported by Hong et al. (2004) for G-area and by Jin et al. (2004) for Z-area. The benchmark dose of Cd required for renal dysfunction to occur was much lower in the heavily As contaminated G-area (Hong et al., 2004) than in the mainly Cd contaminated Z-area (Jin et al., 2004). In addition to the observations concerning interactions, it is interesting that in area G, where arsenic exposure was most prominent, effects on albumin excretion, a biomarker of glomerular dysfunction was most prevalent. It has not previously been clearly demonstrated in humans that environmental exposures to arsenic give rise to renal glomerular dysfunction. It is most probable that Asexposures in G-area are predominantly inorganic As. However, the determinations of UAs were only for total Table 5 Relationship between UCd, UIAs and RBP, B2M among women in Z-area (Data from Buchet et al., 2003) UCd, UIAs and RBP and B2M in women living in Z area
UCd b 4.8 Ag/gCr UCd N 4.8 Ag/gCr a
UIAs (Ag/gCr).
As As As As
b N b N
36a 36a 36a 36a
N
RBP (mg/gCr)
B2M (mg/gCr)
124 87 75 130
0.059 0.083 0.077 0.163
0.140 0.189 0.202 0.344
As. Since G-area is an inland area where consumption of fish and shellfish is very low, and exposure to inorganic As is high, it is still reasonable to assume that UAs values reflect mainly inorganic As. Renal tubular toxicity has been well documented previously in humans exposed for a long time to environmental Cd (Jarup et al., 1998; Jin et al., 2004; Nordberg, 2004; Nordberg et al., 2002). In the present study, renal tubular dysfunction, indicated by a high prevalence of elevated excretion of NAG and B2M, occurred in both Gand Z-areas among persons with high exposure to Cd and low exposure to As. It is interesting to note that also among persons with predominant As-exposure (and low exposure to Cd), there was increased prevalence of renal tubular dysfunction biomarkers (increased UB2M and UNAG). Such a relationship between As exposure and renal tubular dysfunction in humans has not been well documented previously. There is a need to confirm the presently reported findings by additional epidemiological studies employing improved methods for determination of As in urine and possibly improved biomarkers of biological damage and dysfunction on a larger sample of the exposed populations.
Conclusions ! The present studies, which employed sensitive biomarkers of renal dysfunction, give support to the idea that human co-exposure to Cd and inorganic As gives rise to more pronounced renal damage than exposure to each element alone. ! Further studies, possibly including additional new biomarkers and improved exposure assessment technology, are needed to firmly establish this interaction.
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Acknowledgments This study was funded by the European Commission INCO-DC programme (No.ERB3514PL971430) and by the National Natural Science Foundation of China (No.30060077/C030108).
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