Effects of low-level arsenic exposure on urinary N -acetyl-β-D-glucosaminidase activity

Abstract

This study was aimed to evaluate whether renal tubular function is impaired by exposure to relatively low concentrations of arsenic. Mean urinary arsenic concentrations and N-acetyl-β-d-glucosaminidase (NAG) activities were compared among 365 and 502 Korean men and women, respectively, in relation to gender, smoking, alcohol consumption, and recent seafood consumption. The study subjects were divided into 4 groups according to urinary NAG activity and seafood consumption prior to urine sampling, and the correlation between arsenic concentration and urinary NAG activity was tested for each group. The mean urinary arsenic level was higher in women, non-smokers, and non-drinkers in comparison to men, smokers, and drinkers, respectively. Individuals who consumed seafood within 3 days prior to urine sampling showed a higher mean urinary arsenic level than those who did not. The correlation between urinary arsenic concentration and NAG activity in urine was significant only in subjects who did not consume seafood within 3 days prior to urine sampling and whose urinary NAG activity was 7.44 U/g creatinine (75th percentile) or higher. The urinary arsenic concentration was a significant determinant of urinary NAG activity in subjects with NAG activity higher than 7.44 U/g creatinine and especially in those who had not consumed seafood recently. These facts suggest that a relatively low-level exposure to inorganic arsenic produces renal tubular damage in humans.

Keywords

arsenic tubular damage dimethylarsinic acid

Introduction

Arsenic is ubiquitously found in soil, air, and water, and the arsenic present in the environment is mainly from natural sources.¹ The main sources of arsenic for humans are drinking water and food.² Inorganic arsenic is the main form of arsenic in water, whereas organic arsenic compounds such as arsenobetaine, arsenocholine, and arsenosugars are present in high concentrations in seafood.^2,3 It has been reported that dimethylarsinic acid (DMA) is also detected in fish, shellfish, and other seafood products at a concentration ranging from 0.001 to 0.307 μg/g fresh weight, which was much lower than that of arsenobetaine (0.18–32.0 μg/g fresh weight).⁴

Generally, inorganic arsenic compounds have higher toxicity than their organic counterparts.⁵ Chronic exposure to inorganic arsenic through drinking water remains problematic worldwide.^6,7 Ingested inorganic arsenic is methylated to monomethylarsonic acid in the human body, and monomethylarsonic acid is in turn reduced to monomethyl arsonous acid. Monomethyl arsonous acid is then methylated to DMA, which is reduced to dimethylarsinous acid. In humans, this process remains incomplete and some arsenic remains as either inorganic arsenic or monomethyl arsenic (MMA). The main urinary metabolite is DMA.^8,9 Chronic exposure to inorganic arsenic via consumption of contaminated drinking water may cause cardiovascular, neurologic, respiratory, hepatic, and dermal diseases as well as cancers of the skin, lung, bladder, or kidney.¹⁰ Previous studies have shown that high level arsenic induces increases in urinary excretion of retinol binding protein and β₂-microglobulin.^11,12 Occupational exposure to arsenic resulted in increasing urinary arsenic levels and produced nephrotoxic effects manifested by higher levels of low molecular weight protein in urine.¹³ Chronic co-exposure to low levels of cadmium and arsenic can cause renal tubular damage.¹⁴ Renal tubular damage increases urinary N-acetyl-β-d-glucosaminidase (NAG) activity and β₂-microglobulin levels.¹⁵ NAG activity is a more sensitive measure than β₂-microglobulin of renal tubular damage caused by chronic exposure to low levels of cadmium or arsenic.¹⁴ We hypothesize that chronic exposure to low to moderate levels of inorganic arsenic can induce renal tubular damage. This study was aimed to identify whether any significant relationship exists between arsenic concentration in urine and NAG activity and also to evaluate if renal tubular function is impaired by low concentrations of arsenic.

Methods

Study subjects

The metal mines density of Chungbuk Province, Republic of Korea, is above the average of the country. This study is a part of an investigation on the heavy metal exposure level of Chungbuk Provincial residents. Target population recruited as subjects for this study were individuals who were 20 years old or older and residing in a rural area of the Province. After listening the purpose, 912 individuals agreed to participate in this study on their free will, and among them, 867 individuals (365 men and 502 women) who provided their written consent and urine samples became the study subjects of this present study. A direct interview was conducted by experienced interviewers, using a standard questionnaire that included questions about demographics, history of smoking, alcohol consumption, drinking water type, and medical and family history of cancer.

The participants were divided into 4 groups and the correlation between arsenic concentration and NAG activity in urine in each group was tested using statistics.

Group 1: Subjects who consumed seafood within 3 days prior to urine sampling and whose urinary NAG activity was lower than 7.44 U/g creatinine (75th percentile).

Group 2: Subjects who did not consume seafood within 3 days prior to urine sampling and whose urinary NAG activity was lower than 7.44 U/g creatinine.

Group 3: Subjects who consumed seafood within 3 days prior to urine sampling and whose urinary NAG activity was 7.44 U/g creatinine or higher.

Group 4: Subjects who did not consume seafood within 3 days prior to urine sampling and whose urinary NAG activity was 7.44 U/g creatinine or higher.

The institutional review board at Chung-Ang University approved this study (approval number, 2007-1; date of proposal, June 21, 2007).

Determination of arsenic in urine

The arsenic concentration in urine was analyzed using an atomic absorption spectrometer atomic absorption spectrometer (AAS) (Perkin-Elmer Model 5100) equipped with a hydride generation system (FIAS 400). Two milliliters of urine sample was mixed with 2 ml of HCl (32%, w/v), 1 ml of ascorbic acid (5%, w/v), and 1 ml of KI (5%, w/v). This mixture was incubated for 1 h and then diluted with 10% HCl. Reducing agents were 0.2% NaBH₄ and 0.5% NaOH; the mobile phase was 3% HCl, and argon was used as the carrier gas.¹⁶ The accuracy of the arsenic concentrations in urine was determined using a commercial standard reference material (Lyphocheck Urine Metal Control, BioRad, USA). The arsenic concentration in the diluted solutions was calculated using external standard curves.

The urinary creatinine level was measured in gram per liter using the Jaffe method,¹⁷ and the unit of urinary arsenic concentration—micrograms per liter—was converted into creatinine units and given as micrograms per gram creatinine.

This method detects inorganic arsenic (arsenite and arsenate) and its organic metabolites (MMA and DMA), which do not include organic arsenicals such as arsenobetaine, arsenocholine, and arsenosugars, and provides a combined measurement value of inorganic arsenic (arsenite and arsenate), MMA and DMA.¹⁴

Activity of NAG in urine

NAG activity was measured using a reaction in which sodium m-cresolsulfon-phthaleniuyl N-acetyl-β-d-glucosaminide was hydrolyzed to N-acetyl-β-d-glucosaminide and m-cresolsulfonphthalein by NAG.¹¹ Quantitative analysis of NAG activity was performed using a commercial kit (Shionogi, Japan) following the manufacturer’s protocol. In short, a synthetic substrate solution (1 ml) was incubated at 37°C for 5 min. The supernatant of the urine samples (50 ml) obtained by centrifuging was mixed with a warm synthetic substrate solution and the mixture was then incubated in a 37°C water bath for 15 min. The stopping solution (2 ml) was then added and mixed thoroughly. The fluorescence intensities of the samples and NAG standard solution were measured at wavelengths of 580 nm, using a spectrophotometer. The unit of urinary NAG activity—units per liter—was corrected with urinary creatinine units and presented as units per gram creatinine.

Statistical analysis

Differences in the mean urinary arsenic concentration were statistically analyzed according to gender, age, history of smoking, alcohol consumption, and recent seafood consumption by using Student’s t-test, and according to the drinking water type by using the ANOVA test. For subjects whose NAG activity was 7.44 U/g creatinine or higher, multivariate analysis was performed using a general linear model to identify determinants of urinary NAG activity.

Results

Demographic characteristics of our study subjects are shown in Table 1.

Table 1.

Geometric means and geometric standard deviations of urinary arsenic level and NAG activity

Variables	N	Urinary arsenic level (μg/g creatinine)	Urinary NAG activity (unit/g creatinine)
All subjects	815	8.47 (1.89)	4.90 (1.93)
Sex
Male	355	7.85 (1.90)^a	4.71 (1.93)
Female	460	8.94 (1.88)	5.00 (1.93)
Age group
−49	121	8.17 (1.80)	3.46 (1.99)^b
50–59	150	8.50 (1.79)	4.18 (1.90)
60–69	206	8.76 (2.01)	5.00 (1.88)
70–	338	8.33 (1.90)	5.87 (1.84)
Mine working history
No	695	8.58 (1.90)	4.81 (1.93)
Yes	119	8.00 (1.84)	5.47 (1.88)
Current smoking status
Non-smokers	632	8.76 (1.88)^b	4.81 (1.92)
Current-smokers	182	7.54 (1.90)	5.16 (1.99)
Alcohol drinking status
Non-drinkers	509	9.03 (1.86)^b	4.85 (1.93)
Drinkers	305	7.61 (1.90)	4.90 (1.95)
Drinking water type
Local water supply	245	8.85 (1.88)	4.71 (1.93)^a
Underground water	333	8.08 (1.93)	4.66 (1.90)
Municipal water supply	127	8.67 (1.75)	5.47 (1.93)
Others	108	8.50 (1.90)	5.53 (2.03)
Seafood intake within 3 days of sampling
No	439	8.00 (1.92)^b	5.10 (1.95)^a
Yes	375	9.12 (1.84)	4.62 (1.90)

NAG: N-acetyl-β-d-glucosaminidase.

^a p < 0.05.

^b p < 0.01.

The geometric mean (geometric standard deviation) of urinary arsenic concentration for all subjects was 8.47 (1.89) μg/g creatinine (Table 1). The mean urinary arsenic levels of women, non-smokers, and non-drinkers were significantly higher than those of men, smokers, and drinkers, respectively. Individuals who consumed seafood within 3 days prior to sampling showed a higher geometric mean of urinary arsenic level than those who did not. In comparisons of mean urinary arsenic level according to age and drinking water type, no statistical difference was observed.

The geometric mean (geometric standard deviation) of urinary NAG activity in all study subjects was 4.90 (1.93) U/g creatinine. Urinary NAG activities of 47 subjects (5.42%) were higher than 11.5 U/L, the upper limit of normal range. Urinary NAG activity showed an increasing tendency as the age of the subjects increased, but no gender difference was found. There was no difference in the geometric means of NAG activity in urine between smokers and non-smokers, and between drinkers and non-drinkers. However, subjects who did not consume seafood within 3 days prior to sampling showed a higher urinary NAG activity than who did, and drinking water type correlated significantly with NAG activity (Table 1). The participants were divided into 4 groups on the basis of recent seafood consumption and urinary NAG activity, and the correlation between urinary arsenic concentration and NAG activity in urine was statistically tested for each group. The correlation coefficient was significant for subjects who did not consume seafood within 3 days prior to urine sampling and whose urinary NAG activity was 7.44 U/g creatinine (75th percentile) or higher. However, no significant correlation between urinary arsenic concentration and NAG activity was found for the other groups (Table 2). In subjects whose NAG activity was lower than 7.44 U/g creatinine, urinary NAG activity was dependent on age. However, the natural logarithm of urinary arsenic concentration was a significant determinant of urinary NAG activity after controlling the effects of age, sex, smoking, and drinking water type in subjects whose NAG activity was 7.44 U/g creatinine or higher and especially in those who had not consumed seafood recently (data not shown).

Table 2.

Correlation coefficients between urinary arsenic level and NAG activity, according to NAG activity group, sex, and seafood intake before sampling

Seafood intake within 3 days before sampling	NAG activity
	Low^a			High^b
	All subjects	Males	Females	All subjects	Males	Females
No	0.052	0.033	0.064	0.293^d	0.270	0.296^c
Yes	−0.171^d	−0.092	−0.024^d	0.114	0.266	0.106

NAG: N-acetyl-β-d-glucosaminidase.

^a Defined as NAG activity of <7.44 U/g creatinine (75percentile value).

^b Defined as NAG activity of ≥7.44 U/g creatinine (75percentile value).

^c p < 0.05.

^d p < 0.01.

Discussion

In this study, we investigated the relationship between urinary arsenic level and NAG activity in individuals who were not occupationally exposed to arsenic. In general, blood urea nitrogen (BUN) and serum creatinine levels have been used to examine renal function. However, these tests yield positive results only after kidney damage by a nephrotoxic agent has progressed.¹⁸ Urinary NAG activity has been used to detect kidney damage before clinical symptoms appear.^15,19 Since NAG is a large molecular weight protein, it cannot pass through intact basement membranes of the glomerulus; consequently, NAG activity in urine is increased if renal microtubules are damaged or in various kidney diseases. Hence, urinary NAG activity has been used to detect microscopic changes in renal tubules before obvious abnormalities in renal function manifest.¹⁵

The geometric mean of the urinary arsenic levels of the control groups in our previous studies, which included those who were not occupationally exposed to arsenic and those who did not live in metal-contaminated areas was 8.36 μg/g creatinine for those who lived in coastal areas and 7.11 µg/g creatinine for those who lived inland. The geometric mean of urinary NAG activity was 2.47 U/g creatinine for the former and 4.83 U/g creatinine for the latter (data not shown). As compared to the subjects of the present study, the controls living inland showed a lower NAG activity but similar urinary arsenic levels, whereas those living in coastal areas showed a similar NAG activity but lower arsenic levels.

In this present study, women had a higher mean urinary arsenic level than males. Women tend to have a higher intake of organic and inorganic arsenic in France,⁴ and in the United States, the urine of women contains a higher proportion of DMA and lower proportions of inorganic arsenic and MMA than that of men.⁹ Another study reported that the geometric mean of creatinine-corrected urinary arsenic concentration is higher in Korean women than in Korean men.¹⁴ Mean urinary arsenic levels were higher for non-smokers and non-drinkers than for smokers and drinkers, respectively. This finding can be explained by the facts that women had a higher mean level of arsenic in urine and the proportion of smokers and drinkers among women was lower than that among men.

The mean urinary arsenic concentration was higher in individuals who consumed seafood within 3 days before sampling, whereas NAG activity in urine was significantly higher in those who did not. Fish and shellfish are the main sources of dietary arsenic, but only 0.4%–5.3% of the arsenic present in seafood is in the inorganic form.^4,20 The organic forms of arsenic—mainly asenobetaine, arsenocholine, and DMA—are predominant in seafood matrices.^20,21 Buchet et al.²² reported an increase in urinary DMA without any changes in the levels of inorganic arsenic and MMA after eating shellfish in different groups of subjects from the general population. Similar increases in DMA associated with seafood intake have also been shown in other studies.^23–25 Arsenosugars abundant in seaweeds, mussels, clams, and oysters are metabolized in human with substantial increase in DMA.²³

The method used to measure urinary arsenic concentration in our study detects inorganic arsenic (arsenite and arsenate) and its organic metabolites (MMA and DMA) but does not detect organic arsenicals such as arsenobetaine, arsenocholine, and arsenosugar. Hence, the main arsenic form that was increased in urine of individuals who consumed seafood might be DMA which was converted from arsenosugars and did not induce renal tubular damage in individuals with no occupational exposure to arsenic.²⁶

The correlation between urinary arsenic concentration and NAG activity in urine was statistically significant in subjects who did not consume seafood within 3 days prior to urine sampling and whose urinary NAG activity was 7.44 U/g creatinine (75th percentile) or higher (Table 2) but not in those with recent seafood intake or with urinary NAG activity lower than 7.44 U/g creatinine. The natural logarithm of urinary arsenic concentration was a significant determinant of urinary NAG activity after controlling the effects of age, sex, smoking, and drinking water type in subjects whose NAG activity was 7.44 U/g creatinine or higher and especially in those who had not consumed seafood recently. Considering that inorganic arsenic is the main exposure form for subjects who did not consume seafood within 3 days prior to urine sampling, it could be suggested that the increase in urinary NAG activity was produced not by exposure to arsenic via seafood intake but by exposure to relatively low levels of inorganic arsenic. An increased prevalence of renal tubular dysfunction biomarkers among persons with predominant arsenic exposure has been reported in a study with subject groups residing in two metal contaminated areas in China.¹² The geometric means of urinary arsenic in the subject groups were 288.40 and 154.03 μg/g creatinine, and those of urinary NAG activity were 11.88 and 8.97 U/g creatinine, respectively. Those values were much higher than the values of the present study (8.47 μg/g creatinine and 4.90 U/g creatinine). This fact suggests that the environmental exposure to inorganic arsenic at much lower level than previously reported can give rise to renal tubular damage in humans. Arsenic-related NAG increase, which did not accompany the increase in the blood urea nitrogen or serum creatinine level, might not be clinically significant, but it suggests microscopic nephrotoxicity induced by exposure to inorganic arsenic. Thus, the level of inorganic arsenic is a more appropriate marker for estimating the risk of nephrotoxicity by arsenic than the total urinary arsenic concentration, including DMA concentration.

We used an AAS method to measure urinary excretion of the sum of inorganic arsenic, MMA, and DMA for biological monitoring of exposure to inorganic arsenic; this method has been adopted by the American Conference of Governmental Industrial Hygienists (ACGIH) and Deutsche Forschungsgemeinschaft (DFG) to measure urinary excretion of arsenic in workers who are occupationally exposed to inorganic arsenic.^27,28 If urinary arsenic concentration measured by this AAS method is used as an exposure biomarker for inorganic arsenic in persons who are not occupationally exposed to arsenic, seafood intake must be avoided for more than 2 days to decrease the urinary DMA to the basal level.²⁹

NAG activity may also increase because of some other toxicants released from mines or chronic diseases such as diabetes. A shortcoming of this study is that the risk factors for kidney disease were not entirely controlled for.

In summary, it is possible that exposure to relatively low levels of inorganic arsenic produces renal tubular damage in humans.

Footnotes

This work was supported by the research grant of the Chungbuk National University in 2008.

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