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Levels of As, Cd, Pb, Cu, Se and Zn in bovine kidneys and livers in Jamaica
ARTICLE IN PRESS Ecotoxicology and Environmental Safety 72 (2009) 564– 571
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Levels of As, Cd, Pb, Cu, Se and Zn in bovine kidneys and livers in Jamaica Jerome Nriagu a,, Mazen Boughanen a, Aaron Linder a, Andrea Howe b, Charles Grant b, Robin Rattray b, Mitko Vutchkov b, Gerald Lalor b a b
Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA International Centre for Environmental and Nuclear Sciences (ICENS), University of the West Indies, Mona Kingston 7, Jamaica
a r t i c l e in f o
a b s t r a c t
Article history: Received 18 August 2006 Received in revised form 26 April 2008 Accepted 1 May 2008 Available online 13 June 2008
Paired liver and kidney samples from 100 free-range cattle in different parts of Jamaica were analyzed for essential and non-essential trace elements. We found significant enrichment of elements in the kidney (K) compared to the liver (L) with the K/L concentration ratios being 5.2 for Cd, 4.1 for Pb, 3.5 for Se and 2.1 for As, but the Cu contents of the kidney were significantly higher with the K/L ratio of 0.45. A large number of kidney and liver samples showed Cu concentrations in the ranges that were associated with deficiency effects in mammals. About 15% of the hepatic samples had Zn concentrations below 20 mg/g, suggesting that there might be zinc insufficiency in some of the animals. Positive associations were found between the metals in both the kidney and liver. On average, the intake of Cd from consumption of both bovine kidney and liver from the island was estimated to be 5.2 mg/day, equivalent to about 7% of the provisional tolerable daily intake (PTDI), although anyone who habitually consumed the few kidneys or livers with 440 mg/g cadmium may be at some risk of exceeding the PTDI. The consumption of offal from local animals did not appear to be an important dietary source of any of the essential microelements. & 2008 Elsevier Inc. All rights reserved.
Keywords: Bovine kidney Bovine liver Copper deficiency Zinc metabolism Inter-element relationships Exposure risk Soil cadmium Essential trace elements Non-essential trace elements
1. Introduction The presence of trace elements in farm animals is of interest from both the animal health and human health perspectives (Bowen, 1978; Nriagu, 1986). Exposure of livestock to either high levels of toxic metals (such as Cd and Pb) or less than optimal levels of the essential microelements (such as Cu, Co and Zn) can engender adverse effects such as reproductive impairment, physiological abnormalities, behavioral modifications or even death (Bedwal et al., 1993; Frank et al., 2000; Tapiero and Tew, 2003; Dorton et al., 2003; Custer et al., 2004; Martelli and Moulis, 2004; Sharma et al., 2005). Metals accumulated in livestock can be passed on to people who consume the meat and can become a health hazard to the public. Of all the animal tissues, kidney and liver constitute a special dilemma in that they have a propinquity to bioaccumulate toxic metals such as Pb, Cd, Hg and As (Alonso et al., 2000, 2002) but can also serve as a rich source of essential microelements (notably Fe, Cu, Zn and Se) in human diet (Vos et al., 1987; Arnold et al., 2006). Kidney and liver are low in cost and are a component of some traditional Jamaican diet. In a recent survey of the dietary habits of 723 adults in the central part of Jamaica (Manchester, St. Elizabeth and Trelawny Parishes), 70% of the respondents said that they do eat liver while 37% claimed that Corresponding author. Fax: +1734 615 7141.
E-mail address: [email protected] (J. Nriagu). 0147-6513/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2008.05.001
they eat kidney (Nriagu, in preparation). A goal of this paper is to determine the levels of Pb, Cd, As, Se, Cu and Zn in kidney and liver samples from cows slaughtered in the country and provide a preliminary assessment of the potential risks and benefits of consuming these organs by the local population. The six elements selected for study (As, Cd, Cu, Pb, Se and Zn) are either known to be elevated in soils of Jamaica (Lalor, 1995; Johnson et al., 1996; Lalor et al., 1998, 1999) or to be physiologically important in livestock. The accumulation of any of these elements in internal organs of cattle and other animals often can be mediated by the absorption and metabolic cycle of the other elements (Abdelrahman and Kincaid, 1992; Bebe and Panemangalore, 1996; Frank et al., 2000; Dorton et al., 2003; Custer et al., 2004; Coudray et al., 2006). This paper explores the inter-element and inter-organ relationships in metal concentrations and the influence of the essential microelements (Zn, Cu and Se) on the accumulation of toxic elements (Cd, Pb and As) in the kidney and liver samples.
2. Methodology 2.1. Sample collection and preparation Paired samples of kidney and liver of 100 animals were obtained from various parts of Jamaica (Fig. 1). The cattle were
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from small farmers who raised their animals mainly on local pasture. The samples were collected from abattoirs by government-employed meat inspectors who received instructions on how to minimize sample handling and the risk of contamination during sample collection. The sample was taken as soon as each organ was removed from a new slaughter. About 100 g of the central lobe of the liver and half of the kidney were collected. Each sample was placed in a zip lock bag and the kidney–liver pair from each cow was put together in a separate zip lock bag. The samples were labeled and transported to the lab where they were frozen and stored. Each inspector was required to complete a form showing the age and gender of each animal, the address of the farm supplying the animal and the date of sample collection. Samples were only collected where the requisite information on the form could be obtained; samples where the farms that supplied the cows were not indicated were excluded from the study. The frozen samples were subsequently thawed at room temperature. The fatty tissue was carefully removed with a stainless steel surgical scalpel and the samples cut into pieces of approximately 1 in. thick and weighed. After oven drying the samples at 80 1C for 2–5 days, the moisture content was measured by weighing again and the sample was ground in an automated agate mortar and pestle. Analyses were carried out on the dried samples but the results are, as usual, quoted on a fresh weight basis. 2.2. Sample digestion Linear polyethylene tubes used in sample digestion were soaked in 5 M nitric acid overnight, rinsed with Milli-Q water (Millipore Corp., Bedford, MA) and then air dried. The sample was directly weighed in the tube and 3 mL of nitric acid (trace metal grade; Fisher, Chicago) was added. The tube and contents were allowed to sit for 20 min at room temperature in a well of a graphite-heating block before the temperature was slowly ramped to 70 1C. The sample was maintained at this temperature until the dissolution of the sample yielded a clear solution. The sample was cooled to room temperature and 2 mL of 30% hydrogen peroxide (supra pure grade, Seastar, Sidney, BC) was added to aid in the complete dissolution of any residual organic matter. The mixture was then heated at 50 1C for approximately 30 min, cooled and diluted to 10 mL with Milli-Q water. 2.3. Instrumental analysis Metal concentrations in the digested samples were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS)
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equipped with a collision cell (Agilent model 7500c; Agilent Technologies, Palo Alto, CA). The instrument was run in the multielement mode (Slotnick et al., 2005). 2.4. Quality assurance A standard reference material (SRM) consisting of bovine liver (SRM 1577b) from the National Institute of Standards and Technology (NIST) was included in each batch of 12 samples analyzed. The ratio of the certified result to the result obtained in each batch was used to calculate the percent recovery for each element. Each batch also included a blank (3 mL of concentrated nitric acid in digestion tube) which was processed exactly like the samples. The method detection limit was estimated as three times the standard deviation of the blank measurement for each element. The blank value for each element was subtracted from the sample results for the particular batch to derive the data presented. 2.5. Data analysis All data were tested for normal distribution (Kolmogorov– Smirnov) and for homogeneity of variance (F-test). The appropriate parametric/non-parametric statistical tests (t-test, analysis of variance—ANOVA) and statistical comparisons (Spearman rank correlation coefficient) were then applied. In addition to a oneway ANOVA, multi-factorial ANOVA was also used to infer interactions between parameters. Calculations of basic statistical parameters (geometric, arithmetic means, etc.) and ANOVA analysis were performed with SPSS (Chicago, IL).
3. Results The recoveries achieved by the analytical method used in the study were within 715% of the certified reference values for all elements except As (Table 1). The recovery for As (145% higher than the value quoted in the SRM) was outside the acceptance (15%) criteria. It should be noted that arsenic in the reference material (0.05 mg/g) was not certified. The higher recovery of this element may be due to two factors: (a) the certified value may not be the true concentration, and (b) arsenic is a difficult element to analyze by ICP-MS and the disparity in values may reflect, at least partially, the uncertainty in the quantification of very low levels of As in the biological materials. The analytical detection limits for the six elements discussed in this paper were 0.28 mg/g for Zn, 0.08 mg/g for Cd, 0.04 mg/g for Cu and 0.01 mg/g for As, Se and Pb (Table 1).
Fig. 1. Map of Jamaica (not drawn to scale) showing the distribution of farm locations where samples used in the study were derived. The parishes are labeled as follows: 1, Hanover; 2, Westmoreland; 3, Saint James; 4, Saint Elizabeth; 5, Trelawny; 6, Manchester; 7, Saint Ann; 8, Clarendon; 9, Saint Catherine; 10, Saint Mary; 11, Saint Andrew; 12, Portland; 13, Saint Thomas.
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Table 1 Quality assurance data from the determination of As, Cd, Cu, Pb, Se and Zn in samples and the standard reference material (N ¼ 10) Material analyzed
Average metal concentrations (mg/g dry wt)
Standard reference material Sample batches Average % recovery Detection limit a
As
Cu
Zn
Se
Cd
Pb
0.05a 0.0770.02 145 0.01
16078.0 15379.6 95.8 0.037
127716 113717 89.1 0.28
0.7370.06 0.64 88.0 0.01
0.5070.03 0.4070.04 85.6 0.08
0.1370.01 0.1170.01 86.2 0.01
Value not certified.
Table 2 Arithmetic averages, geometric means and ranges in concentrations of Pb, Cd, As, Zn, Cu and Se in bovine kidney and liver samples in mg/g dry weight (wet weight in parentheses) Element
Liver (N ¼ 100) Averagea
Pb Cd As Zn Cu Se
GMa b
0.162 (0.052) 10.1 (3.24) 0.050 (0.016) 92.2 (29.5) 63.9 (20.4) 1.35 (0.432)
K/L ratioc
Kidney (N ¼ 100)
0.071 1.18 0.027 86.9 31.5 0.974
(0.023) (0.378) (0.009) (27.8) (10.1) (0.312)
Range
Averagea
GMa
Range
BDL–1.30 (BDL–0.416) BDL–256 (BDL–82.1) BDL–1.12 (BDL–0.358) 16.6–229 (5.28–73.6) BDL–312 (BDL–99.8) 0.180–8.48 (0.058–2.71)
0.523 (0.126) 33.1 (7.92) 0.106 (0.025) 84.8 (20.4) 16.2 (3.89) 4.23 (1.02)
0.292 (0.070) 6.17 (1.48) 0.056 (0.013) 79.3 (19.0) 14.0 (3.36) 3.44 (0.825)
0.017–3.57 (0.004–0.857) 0.052–487 (0.012–117) 0.004–1.36 (0.001–0.326) 13.2–188 (3.17–45.2) BDL–91.2 (BDL–21.9) BDL–14.9 (BDL–3.58)
4.1 5.2 2.1 0.91 0.45 3.5
Note: average blank value was subtracted from each individual sample value. a Average: arithmetic average; GM: geometric mean; BDL: below the instrumental detection limit shown in Table 1. b Values in parentheses are in wet weight. c K/L is the ratio of average concentration in the kidney to average concentration in liver samples.
The geometric means (GM), arithmetic averages and ranges in concentrations of Pb, Cd, As, Zn, Cu and Se in the kidney and liver samples both on wet-weight and dry-weight basis are shown in Table 2. Measured average water contents for the kidney and liver samples were 7675.5% and 6872.4%, respectively. To convert the concentrations given for dry-weight to wet-weight basis, the values should be multiplied by 0.24 for the kidney and 0.32 for liver samples to account for the water contents of these organs. We explored the influence of age and gender on the concentrations of the trace metals using multiple linear regression models. Age was only found to be a predictor of Cd (t ¼ 4.38; po0.001) in the kidney and was not significantly associated with any element in the liver (Table 3). The Cd contents of older cows were much higher than those of younger animals. Gender was a predictor of the Cu in kidneys of the test animals (t ¼ 3.01; p ¼ 0.004) with levels in female animals being higher than those of male cows. Female cows also had more Se in their livers than the males (t ¼ 2.39; p ¼ 0.019) (Table 3). Non-parametric Spearman correlation was performed to examine the inter-element relationships in cattle organs and the results are presented in Table 4. For liver samples, weak positive associations were found between copper and zinc (p ¼ 0.044, r ¼ 0.198), as well as zinc and lead (p ¼ 0.016, r ¼ 0.239); moderate positive associations was shown between cadmium and zinc (p ¼ 0.0002, r ¼ 0.361); while a moderate negative association between copper and arsenic (p ¼ 0.0009, r ¼ 0.327) was found (Table 4). The kidney samples showed a very strong positive correlation between cadmium and zinc (po0.0001, r ¼ 0.70); a positive correlation between copper and zinc (p ¼ 0.019, r ¼ 0.233), arsenic and selenium (p ¼ 0.013, r ¼ 0.247) and lead and selenium (p ¼ 0.007, r ¼ 0.269). The inter-organ relationships were compared for each metal (Table 4) and only weak positive associations were detected for cadmium (p ¼ 0.019, r ¼ 0.235) and selenium (p ¼ 0.015, r ¼ 0.243).
Table 3 Multiple linear regression of age and gender versus (log transformed) concentrations of trace metals in kidney and liver samples Cd Age (kidney) t 4.38 p-value o0.001
Cu
Pb
Zn
Se
As
1.30 0.197
1.26 0.211
1.07 0.287
0.799 0.427
0.571 0.570
1.46 0.149
1.35 0.182
0.890 0.377
1.15 0.256
0.256 0.799
0.605 0.547
Gender (kidney) t 0.493 p-value 0.624
3.01 0.004
0.146 0.884
0.911 0.365
1.39 0.116
1.53 0.131
1.58 0.117
0.463 0.645
0.301 0.764
2.39 0.019
0.088 0.930
Age (liver) t p-value
Gender (liver) t p-value
0.068 0.946
4. Discussion The ratio of average metal concentration in kidney to that in liver (K/L ratio) was highest for Cd (5.2) and was elevated for Pb (4.1), Se (3.5) and As (2.1) (estimated from data in Table 2). By contrast, Cu is depleted in the kidney relative to the liver (K/L ratio ¼ 0.45) and there does not appear to be a preferential accumulation of Zn in the kidney relative to the liver (K/L ratio ¼ 0.91). 4.1. Deficiency/toxicity of the trace metals The geometric mean concentration of Pb in the liver samples (0.07 mg/g wet weight (ww)) falls in the range of 0.05–0.40 mg/g ww that have been reported in many countries (Alonso et al., 2000). Our geometric mean value for Pb in the liver (0.02 mg/g ww), however, is less than the 0.05–0.46 mg/g found in previous
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Table 4 Spearman rank correlation coefficients for log metal concentrations in the animal tissues (N ¼ 100) Cadmium (Cd) Liver samples Cadmium (Cd)
Copper (Cu)
Zinc (Zn)
Arsenic (As)
Selenium (Se)
Lead (Pb)
r ¼ 0.127 p ¼ 0.208
r ¼ 0.361 p ¼ 0.0002
r ¼ 0.126 p ¼ 0.213
r ¼ 0.138 p ¼ 0.171
r ¼ 0.033 p ¼ 0.745
r ¼ 0.198 p ¼ 0.044
r ¼ 0.327 p ¼ 0.0009
r ¼ 0.131 p ¼ 0.197
r ¼ 0.079 p ¼ 0.435
r ¼ 0.030 p ¼ 0.766
r ¼ 0.031 p ¼ 0.759
r ¼ 0.239 p ¼ 0.016
r ¼ 0.115 p ¼ 0.257
r ¼ 0.56 p ¼ 0.582
Copper (Cu)
r ¼ 0.127 p ¼ 0.208
Zinc (Zn)
r ¼ 0.361 p ¼ 0.0002
r ¼ 0.198 p ¼ 0.044
Arsenic (As)
r ¼ 0.126 p ¼ 0.213
r ¼ 0.327 p ¼ 0.0009
r ¼ 0.030 p ¼ 0.766
Selenium (Se)
r ¼ 0.138 p ¼ 0.171
r ¼ 0.131 p ¼ 0.197
r ¼ 0.031 p ¼ 0.759
r ¼ 0.115 p ¼ 0.257
Lead (Pb)
r ¼ 0.033 p ¼ 0.745
r ¼ 0.079 p ¼ 0.435
r ¼ 0.239 p ¼ 0.016
r ¼ 0.560 p ¼ 0.582
r ¼ 0.609 p ¼ 0.549
r ¼ 0.024 p ¼ 0.804
r ¼ 0.700 po0.0001
r ¼ 0.047 p ¼ 0.645
r ¼ 0.023 p ¼ 0.819
r ¼ 0.155 p ¼ 0.122
r ¼ 0.233 p ¼ 0.019
r ¼ 0.141 p ¼ 0.163
r ¼ 0.079 p ¼ 0.436
r ¼ 0.006 p ¼ 0.950
r ¼ 0.019 p ¼ 0.854
r ¼ 0.111 p ¼ 0.273
r ¼ 0.154 p ¼ 0.125
r ¼ 0.247 p ¼ 0.013
r ¼ 0.137 p ¼ 0.176
Kidney samples Cadmium (Cd)
r ¼ 0.609 p ¼ 0.549
Copper (Cu)
r ¼ 0.024 p ¼ 0.804
Zinc (Zn)
r ¼ 0.700 po0.0001
r ¼ 0.233 p ¼ 0.019
Arsenic (As)
r ¼ 0.046 p ¼ 0.645
r ¼ 0.141 p ¼ 0.163
r ¼ 0.019 p ¼ 0.854
Selenium (Se)
r ¼ 0.023 p ¼ 0.819
r ¼ 0.079 p ¼ 0.436
r ¼ 0.111 p ¼ 0.273
r ¼ 0.247 p ¼ 0.013
Lead (Pb)
r ¼ 0.155 p ¼ 0.122
r ¼ 0.006 p ¼ 0.950
r ¼ 0.154 p ¼ 0.125
r ¼ 0.137 p ¼ 0.176
r ¼ 0.269 p ¼ 0.007
r ¼ 0.235 p ¼ 0.019
r ¼ 0.039 p ¼ 0.696
r ¼ 0.098 p ¼ 0.332
r ¼ 0.012 p ¼ 0.905
r ¼ 0.243 p ¼ 0.015
Inter-tissue relationships Kidney–liver metal correlation
r ¼ 0.269 p ¼ 0.007
r ¼ 0.037 p ¼ 0.715
po0.05. po0.01. po0.001.
studies in other countries (Alonso et al., 2000). The low average values are consistent with the fact that neither the liver nor the kidney is an accumulator organ for Pb in mammals (Kramer et al., 1983; Salisbury et al., 1991; Alonso et al., 2000). Concentrations of As in the bovine kidney (o0.015–0.07 mg/g) and liver (o0.015–0.05 mg/g) reported in the literature (Kramer et al., 1983; Vos et al., 1987; Jorhem et al., 1991; Salisbury et al., 1991) are somewhat higher than our geometric mean values of 0.013 mg/ g ww for kidney and 0.01 mg/g ww for liver. Our mean Cd concentrations in the liver (0.38 mg/g ww) and kidney (1.5 mg/g ww) are much higher than the 0.03–0.12 mg/g ww for livers and 0.07–0.65 mg/g ww for kidneys that have been reported in several countries (Nriagu, 1981; Alonso et al., 2000). The enrichment of Jamaican samples with Cd can be expected considering the elevated levels of this element that have been reported in local soils (Engel et al., 1996; Lalor, 1995). Any comparison of our data with those of other studies must be made in a circumspect manner because of differences in analytical methodologies used, physiological conditions of the cows (whether lactating, pregnant
or sickly), age classification of the animal population and the nature of the feed (Olsson et al., 2001; Wlostowski et al., 2006). Reports in the scientific literature (Kramer et al., 1983; Salisbury et al., 1991) typically show Se concentrations of 0.23–0.31 for bovine livers and 0.92–1.5 mg/g for bovine kidneys. These values are consistent with our geometric mean values of 0.82 mg/g ww for the kidney and 0.31 mg/g ww for liver samples (Table 2). According to the criteria developed by Puls (1994), Se would be considered to be deficient (o0.54 mg/g dw) in 28% of the liver samples from our study animals, marginally deficient (0.58–0.86 mg/g dw) in 15% of the samples, adequate to high (0.86–4.24 mg/g dw) in 50% and at chronic toxicity level (44.3 mg/g dw) in 5% of the liver samples. The geometric mean renal Cu concentration in Jamaican bovine samples (3.4 mg/g) is among the lowest reported for cattle in any part of the world (see Alonso et al., 2000; Miranda et al., 2005). Also, the geometric mean hepatic Cu concentration of 10 mg/g found in our study is well below average values of 23–137 mg/g ww that have been reported for cattle in other parts
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of the world (Alonso et al., 2000; Miranda et al., 2005). Two-thirds of the animals studied in this project showed hepatic Cu values below 25 mg/g considered to be normal for cattle (Puls, 1994). Copper concentrations in 48% of the liver samples were below 34 mg/g dry weight (dw) considered to be the deficiency threshold while 26% of the renal values were in the range of 34–85 mg/g dw considered to be the marginally deficient level (Puls, 1994). Interestingly, Cu levels in 52% of the renal samples were o12 mg/g ww found in livers of moose showing ‘‘moose wasting disease’’ associated with Cu deficiency in Alaska, Sweden and Nova Scotia (Frank et al., 2000; O’Hara et al., 2001; Custer et al., 2004; Gamberg et al., 2005). Liver Cu concentration is used to diagnose Cu deficiency in livestock (National Research Council, 1984) and our results for a large number of cows on the island would seem to be in the less than optimal range. Copper deficiency is associated with a number of physiological and biochemical changes including reduced appetite, growth and immune function impairment and increased susceptibility to disease (Underwood, 1971; Dorton et al., 2003). Copper deficiency also impairs normal metabolism, resulting in microcytic, hypochromic anemia and associated hepatic Fe overload, altered structure and functioning of circulating and immune cells, aberrant cardiac electrophysiology and persisitent effects on neurobehavior and the immune system (Uriu-Adams and Keen, 2005; Auclair et al., 2006). There is nothing to indicate that the cattle population in Jamaica is experiencing any of these adverse health effects associated with Cu deficiency, but then the health status of farm animals on the island is not well documented. Copper deficiency is now recognized to be a common problem in many domesticated and wild animals in many countries (UriuAdams and Keen, 2005; Blanco-Penedo et al., 2006), and Jamaica should be added to the list. Copper is commonly supplemented to most feedlot diets in the US and other countries (Dorton et al., 2003), in recognition of the fact free range foraging (a-la in Jamaica) does not provide cattle with adequate supply of this essential microelement. The geometric mean Zn concentration for kidneys (19 mg/g ww) falls within the range of 14–30 mg/g ww found in other studies and within the range considered adequate for cattle tissue (Puls, 1994). The mean value for livers (28 mg/g), however, is lower than 39–70 mg/g reported in many countries (Alonso et al., 2000; Miranda et al., 2005). About 15% of the hepatic samples had Zn concentrations below 20 mg/g and the question of Zn insufficiency in some of theses animals may be raised. Low liver Zn levels in cattle have been associated with exposure to high environmental Cd pollution (Miranda et al., 2005) and the low hepatic Zn concentrations in our samples may be related to high levels of Cd in local soils. About 70% of the variance in hepatic Cd concentrations in our samples (Table 4) may reflect the effects of Zn metabolism or other determinants of Zn concentration in the kidney. The levels of Cd in Jamaican soils may be related to low Cu concentrations in livestock tissues since other studies have reported hypocupraemia in areas where soils are polluted with Cd (Koh and Judson, 1986; Spierenburg et al., 1988; Wentink et al., 1988; Prankel et al., 2005). Whereas deficiencies in essential micronutrients (Cu and Zn) may be an issue, the bioaccumulations of non-essential metals in kidneys and livers may reach levels of concern toxicologically (Nriagu, 1986). For mammals, the threshold levels of biological effects for Cd have been reported to be 20–200 mg/g in the liver and 50–400 mg/g in the kidney (Fisk et al., 2005). Only two liver and two kidney samples from our test animals fell into these ranges. The kidney, liver, lung, heart and testis are the main target organs following Cd exposure and the residence times of Cd in these animal tissues are years-to-decades long (Saratug et al.,
2003). Because of the long retention times and the fact that Cd is not physiologically regulated in mammals, sustained environmental exposures often result in significant accumulation of Cd in these organs in a time-dependent manner. Many previous studies, like ours, have found significant associations between renal and hepatic Cd concentrations with age for a wide variety of organisms including humans (Komarnicki, 2000; Olsson et al., 2001; Saratug et al., 2003; Gamberg et al., 2005).
4.2. Inter-element relationships Because of the role of the liver and kidney in the metabolism of trace metals and the susceptibility of these organs to metal accumulation and toxicity (Underwood, 1971; Taylor, 1996; Alonso et al., 2004), the inter-element relationships in these tissues are generally of concern and scientific interest. Significant interelement relationships provide some indication of the moderating effects on observed tissue accumulations of the metals. The strongest positive interaction observed in our samples was between Cd and Zn in the kidney (po0.0001, r ¼ 0.700) and liver (p ¼ 0.0002, r ¼ 0.361) (Table 4). Statistically significant associations have been likewise reported previously between Zn and Cd (as well as Cu, Pb and other trace metals) in various mammalian tissues (Bebe and Panemangalore, 1996; Taylor, 1996; Frank et al., 2000; Komarnicki, 2000; Coudray et al., 2006; Phillips et al., 2005; Reeves and Chaney, 2004). The liver and a number of tissues contain an ubiquitous Zn binding protein, metallothioneins (MTs), which have at least two function—to sequester Zn and release it by events that signal its requirement (Tapiero and Tew, 2003). These proteins also bind Cd exquisitely. Besides a role in metal homeostasis, the MTs can also serve as store for Zn and Cd, and be involved in metal transfer (Sharma et al., 2005; Phillips et al., 2005). The mechanisms by which Cd affects Zn metabolism are not yet well understood but the strong association of Cd and Zn in the liver and kidney most likely is related to the fact that the handling of Cd2+ and Zn2+ in these tissues intersect at several points. Subchronic dietary exposure to Cd has been linked with collateral increases in Zn and Cu concentrations in the liver and kidney (Phillips et al., 2005). It is conceivable that the significant correlation between Zn and Cu concentrations in the liver (p ¼ 0.044, r ¼ 0.198) and kidney (p ¼ 0.019, r ¼ 0.233) observed in our samples may stem from the indirect effect of co-exposure to high levels of Cd in local soils. The associations may also be related to the sharing of transport systems by the metals and the regulatory effects of MTs which can be independently expressed by any of these three metals. Besides the bones which are the main target organ for Pb accumulation in vertebrates, chronic exposure often results in elevated levels of this metal in the kidney compared to the liver (Ma, 1989; Komarnicki, 2000). The fact that the liver is not accumulator sites for Pb is suggested by the low hepatic Pb level (0.07 mg/g) and high K/L ratio (of 44) found in our samples (Table 2). We found no significant association between the amounts of Pb in the kidney and liver (Table 4), suggesting that the lead pools in these tissues may be unrelated to each other. A number of studies have reported positive correlations between hepatic Zn and Pb concentrations at relative low levels of exposure (Alonso et al., 2002; Rahil-Khazen et al., 2002) while others have found no association (Szefer et al., 1994; Alonso et al., 2004). Although other studies have also reported significant associations between Pb and Zn, Pb and Cu, and Pb and Cd in the kidney (August et al., 1989; Alonso et al., 2002, 2004; RahilKhazen et al., 2002; Miranda et al., 2005), we did not find such correlations in Jamaican samples (Table 4).
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At high doses of exposure, As is said to have a detoxifying effect against Se and vice versa (Bedwal et al., 1993). At low levels of exposure, cells show less selectivity towards selenate and arsenate ions so that a deficiency in one often engenders increased uptake of the other in the liver and kidney (Nriagu, 1994; Miyazaki et al., 2005). The significant positive association between the renal As and Se concentrations (p ¼ 0.013, r ¼ 0.247) in our samples is consistent with sympathetic accumulation of As and Se when the pools of these elements are low in the liver.
4.3. Possible health risks of trace metal levels found in kidney and liver Kidney and liver represent a significant vector for trace metal exposure in some segments of the population of Jamaica (Lalor et al., 2004). Consumption of kidney and liver also represents a significant source of vitamins, proteins, lipids, antioxidants, essential micronutrients and calorific intake for the island population. The nutritional benefits can be substantial and must therefore be weighed against the potential health risks associated with toxic metals that have accumulated in these organs. The goal of this section is not to provide a detailed analysis of the weight of evidence and scientific information on risk and benefits of offal consumption. Since data required for comprehensive risk assessment is lacking, the aim of this section rather is to draw attention and encourage further investigation of potential health effects associated with exposure to As, Cd, Cu, Pb, Se and Zn found in kidneys and livers of locally raised cows. A number of countries have set maximum residue levels (MRL) for these organs to protect human health. There are currently no such guidelines for metal concentrations in animal meats and organs sold in Jamaica and the values for a number of countries
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are listed in Table 5. Whether the guidelines for other countries are valid for Jamaican population is a matter for conjecture at this time. The foreign guidelines are used here merely as a convenient benchmark for discussing our data. With the exception of Cd, the geometric mean concentrations of trace elements measured in liver and kidney of Jamaican cattle are below the MRLs that have been set in many countries (Table 5). When the distributions of individual elements are considered, About 60% of the kidney samples showed Cd values above 1.0 mg/g while 40% of the liver samples had Cd concentrations above 0.5 mg/g—MRL in many countries (Table 5). No sample had Cu, Pb, Zn or As content that was above the recommended guidelines for either the kidney or liver. It should be emphasized that the MRLs in themselves cannot guarantee low-risk exposures (they do not address eating habits, smoking, environmental sources and nutritional factors; Berti et al., 1998) and are used as an expedient tool because they are measurable and enforceable, in principle. Dietary data were recently collected from 723 adults in the central part of the island using food frequency questionnaire (Nriagu, in preparation). Average consumption rates for kidney and liver calculated from this survey were 2.2 and 5.2 g/day, respectively. From current data on hepatic and renal metal concentrations, the average intake of each metal from consumption of the two tissues has been estimated and shown in Table 6. The mean intake of Cd from consumption of both kidney and liver is estimated to be 5.2 mg/day, equivalent to about 7% of the provisional tolerable daily intake (PTDI) value of 72 mg/day recommended by WHO (1989). It should, however, be noted that about 5% of the liver samples analyzed had over 40 mg/g Cd while 15% of the kidney samples had over 40 mg/g Cd. Anyone who habitually consumes kidneys or livers with such elevated levels of Cd may be at some risk of exceeding the PTDI. From our
Table 5 Maximum residue limits (mg/g wet weight) for arsenic, cadmium, lead, copper, zinc and selenium for bovine liver and kidney in various countries and organizations Liver
Kidney
Country
Reference
Lead
0.5 0.5 1 0.6 2
0.5 0.5 1 1.0 1
World Health Organization European Commission Slovac Republic Russia United Kingdom
WHO (2001) EU Commission (1997, 2001) Kottferova and Korenekova (1995) Farmer and Farmer (2000) UK Minisitry of Agriculture, Fisheries and Food (1998)
Cadmium
0.5–1 0.5 0.5 1 0.1
1 0.5 1 1 0.3
European Commission Germany Slovac Republic Australia Russia
EU Commission (1997, 2001) Kreuzer et al. (1988) Kottferova and Korenekova (1995) Kramer et al. (1983) Farmer and Farmer (2000)
Arsenic
1 2 2
1 2 2
Australia Canada World Health Organization
Kramer et al. (1983) Salisbury et al. (1991) WHO (1992)
Copper
100 100 150 60
100 100 150 60
United States Australia Canada Slovac Republic
NAS (2000) Langlands et al. (1988) Salisbury et al. (1991) Kottferova and Korenekova (1995)
Zinc
500 500 150 100 80 100
500 500 150 100 80 100
United States Canada Australia Canada Slovac Republic Russia
NAS (2000) National Research Council (1984) Langlands et al. (1988) Salisbury et al. (1991) Kottferova and Korenekova (1995) Farmer and Farmer (2000)
2 2
2 2
United States World Health Organization
NAS (2000) WHO (1989)
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Table 6 Estimated exposure to trace metals from consumption of kidney and liver compared to the provisional tolerable daily intake (PTDI) for toxic metals or recommended dietary allowance (RDA) for the essential trace elements Element
Average daily consumption (mg/day)
Established guideline
Kidney
Liver
Kidney+liver
Arsenic Cadmium Copper
0.03 3.26 7.39
0.45 1.96 52.4
0.48 5.22 59.79
Lead Selenium Zinc
0.154 1.82 41.9
0.118 1.62 145
0.272 3.44 186.9
results, it would appear that kidneys and livers of a small number of cattle reared in Jamaica may be a health risk to some consumers. It must be emphasized that many people on the island do not eat either kidney or liver and that the risk of exposure to Cd in offal depends on dietary habits, nutritional factors, and presence of diabetic/kidney diseases, smoking, consumption of other types of foods that contain elevated levels of Cd, gender and genetic diseases (Jarup et al., 1998; Meeus et al., 2002). The mean intakes of Pb (0.27 mg/day), As (0.48 mg/day) and Se (3.4 mg/day) from kidney and liver (Table 6) make up o1%, o1% and 7% of the PTDI, respectively; offal represents an insignificant route of exposure for these elements. In terms of the essential trace metals, an average daily consumption of kidney and liver yields 0.98 mg Cu, well below the recommended daily allowance of 9 mg/day in the United States (Johnson et al., 1992; Uriu-Adams and Keen, 2005). The daily intake of Zn from kidney and liver is 0.18 mg, also well below the RDA value of 8 mg/day for women and 11 mg/day for men in the USA (IOM, 2001). The consumption of offal from local animals thus does not appear to be an important dietary source of either Cu or Zn for people on the island.
5. Conclusions The accumulation of trace metals in kidneys and livers of cows in Jamaica is enigmatic. On the one hand, the data suggest that many cattle may be experiencing Cu and, to a lesser extent, Zn deficiency. One consequence of the low Cu and Zn contents is that consumption of offal from local cows does not represent a significant dietary source of the two essential microelements to the local population. On the other hand, Cd levels are elevated in these organs and consumption of large quantities of the offal represents a potential health hazard. There is clearly a need for additional studies to assess the concatenated effects of exposing local cattle to suboptimal amounts of Zn and Cu and elevated doses of Cd.
Acknowledgments Sample collection and preparation were done while the lead author held a senior Fulbright Fellowship at the International Center for Environmental & Nuclear Sciences, University of the West Indies, and financial support for the work is acknowledged. Appreciation is extended to Ms. Myriam Afeiche for assistance with data analysis. Human subjects: The research did not involve any human subjects, hence there was no need for institutional review board (IRB) approval.
PTDI ¼ 154 mg/day (WHO, 2002) PTDI ¼ 72 mg/day (WHO, 2002) RDA ¼ 0.9 mg/day (IOM, 2001) PTDI ¼ 10 mg/day (IOM, 2002) PTDI ¼ 257 mg/day (WHO, 2002) RDA ¼ 55 mg/day (NAS, 2000; ATSDR, 2003) RDA ¼ 8 mg/day for women and 11 mg/day for men (IOM, 2000)
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Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Levels of As, Cd, Pb, Cu, Se and Zn in bovine kidneys and livers in Jamaica Jerome Nriagu a,, Mazen Boughanen a, Aaron Linder a, Andrea Howe b, Charles Grant b, Robin Rattray b, Mitko Vutchkov b, Gerald Lalor b a b
Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA International Centre for Environmental and Nuclear Sciences (ICENS), University of the West Indies, Mona Kingston 7, Jamaica
a r t i c l e in f o
a b s t r a c t
Article history: Received 18 August 2006 Received in revised form 26 April 2008 Accepted 1 May 2008 Available online 13 June 2008
Paired liver and kidney samples from 100 free-range cattle in different parts of Jamaica were analyzed for essential and non-essential trace elements. We found significant enrichment of elements in the kidney (K) compared to the liver (L) with the K/L concentration ratios being 5.2 for Cd, 4.1 for Pb, 3.5 for Se and 2.1 for As, but the Cu contents of the kidney were significantly higher with the K/L ratio of 0.45. A large number of kidney and liver samples showed Cu concentrations in the ranges that were associated with deficiency effects in mammals. About 15% of the hepatic samples had Zn concentrations below 20 mg/g, suggesting that there might be zinc insufficiency in some of the animals. Positive associations were found between the metals in both the kidney and liver. On average, the intake of Cd from consumption of both bovine kidney and liver from the island was estimated to be 5.2 mg/day, equivalent to about 7% of the provisional tolerable daily intake (PTDI), although anyone who habitually consumed the few kidneys or livers with 440 mg/g cadmium may be at some risk of exceeding the PTDI. The consumption of offal from local animals did not appear to be an important dietary source of any of the essential microelements. & 2008 Elsevier Inc. All rights reserved.
Keywords: Bovine kidney Bovine liver Copper deficiency Zinc metabolism Inter-element relationships Exposure risk Soil cadmium Essential trace elements Non-essential trace elements
1. Introduction The presence of trace elements in farm animals is of interest from both the animal health and human health perspectives (Bowen, 1978; Nriagu, 1986). Exposure of livestock to either high levels of toxic metals (such as Cd and Pb) or less than optimal levels of the essential microelements (such as Cu, Co and Zn) can engender adverse effects such as reproductive impairment, physiological abnormalities, behavioral modifications or even death (Bedwal et al., 1993; Frank et al., 2000; Tapiero and Tew, 2003; Dorton et al., 2003; Custer et al., 2004; Martelli and Moulis, 2004; Sharma et al., 2005). Metals accumulated in livestock can be passed on to people who consume the meat and can become a health hazard to the public. Of all the animal tissues, kidney and liver constitute a special dilemma in that they have a propinquity to bioaccumulate toxic metals such as Pb, Cd, Hg and As (Alonso et al., 2000, 2002) but can also serve as a rich source of essential microelements (notably Fe, Cu, Zn and Se) in human diet (Vos et al., 1987; Arnold et al., 2006). Kidney and liver are low in cost and are a component of some traditional Jamaican diet. In a recent survey of the dietary habits of 723 adults in the central part of Jamaica (Manchester, St. Elizabeth and Trelawny Parishes), 70% of the respondents said that they do eat liver while 37% claimed that Corresponding author. Fax: +1734 615 7141.
E-mail address: [email protected] (J. Nriagu). 0147-6513/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2008.05.001
they eat kidney (Nriagu, in preparation). A goal of this paper is to determine the levels of Pb, Cd, As, Se, Cu and Zn in kidney and liver samples from cows slaughtered in the country and provide a preliminary assessment of the potential risks and benefits of consuming these organs by the local population. The six elements selected for study (As, Cd, Cu, Pb, Se and Zn) are either known to be elevated in soils of Jamaica (Lalor, 1995; Johnson et al., 1996; Lalor et al., 1998, 1999) or to be physiologically important in livestock. The accumulation of any of these elements in internal organs of cattle and other animals often can be mediated by the absorption and metabolic cycle of the other elements (Abdelrahman and Kincaid, 1992; Bebe and Panemangalore, 1996; Frank et al., 2000; Dorton et al., 2003; Custer et al., 2004; Coudray et al., 2006). This paper explores the inter-element and inter-organ relationships in metal concentrations and the influence of the essential microelements (Zn, Cu and Se) on the accumulation of toxic elements (Cd, Pb and As) in the kidney and liver samples.
2. Methodology 2.1. Sample collection and preparation Paired samples of kidney and liver of 100 animals were obtained from various parts of Jamaica (Fig. 1). The cattle were
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from small farmers who raised their animals mainly on local pasture. The samples were collected from abattoirs by government-employed meat inspectors who received instructions on how to minimize sample handling and the risk of contamination during sample collection. The sample was taken as soon as each organ was removed from a new slaughter. About 100 g of the central lobe of the liver and half of the kidney were collected. Each sample was placed in a zip lock bag and the kidney–liver pair from each cow was put together in a separate zip lock bag. The samples were labeled and transported to the lab where they were frozen and stored. Each inspector was required to complete a form showing the age and gender of each animal, the address of the farm supplying the animal and the date of sample collection. Samples were only collected where the requisite information on the form could be obtained; samples where the farms that supplied the cows were not indicated were excluded from the study. The frozen samples were subsequently thawed at room temperature. The fatty tissue was carefully removed with a stainless steel surgical scalpel and the samples cut into pieces of approximately 1 in. thick and weighed. After oven drying the samples at 80 1C for 2–5 days, the moisture content was measured by weighing again and the sample was ground in an automated agate mortar and pestle. Analyses were carried out on the dried samples but the results are, as usual, quoted on a fresh weight basis. 2.2. Sample digestion Linear polyethylene tubes used in sample digestion were soaked in 5 M nitric acid overnight, rinsed with Milli-Q water (Millipore Corp., Bedford, MA) and then air dried. The sample was directly weighed in the tube and 3 mL of nitric acid (trace metal grade; Fisher, Chicago) was added. The tube and contents were allowed to sit for 20 min at room temperature in a well of a graphite-heating block before the temperature was slowly ramped to 70 1C. The sample was maintained at this temperature until the dissolution of the sample yielded a clear solution. The sample was cooled to room temperature and 2 mL of 30% hydrogen peroxide (supra pure grade, Seastar, Sidney, BC) was added to aid in the complete dissolution of any residual organic matter. The mixture was then heated at 50 1C for approximately 30 min, cooled and diluted to 10 mL with Milli-Q water. 2.3. Instrumental analysis Metal concentrations in the digested samples were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS)
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equipped with a collision cell (Agilent model 7500c; Agilent Technologies, Palo Alto, CA). The instrument was run in the multielement mode (Slotnick et al., 2005). 2.4. Quality assurance A standard reference material (SRM) consisting of bovine liver (SRM 1577b) from the National Institute of Standards and Technology (NIST) was included in each batch of 12 samples analyzed. The ratio of the certified result to the result obtained in each batch was used to calculate the percent recovery for each element. Each batch also included a blank (3 mL of concentrated nitric acid in digestion tube) which was processed exactly like the samples. The method detection limit was estimated as three times the standard deviation of the blank measurement for each element. The blank value for each element was subtracted from the sample results for the particular batch to derive the data presented. 2.5. Data analysis All data were tested for normal distribution (Kolmogorov– Smirnov) and for homogeneity of variance (F-test). The appropriate parametric/non-parametric statistical tests (t-test, analysis of variance—ANOVA) and statistical comparisons (Spearman rank correlation coefficient) were then applied. In addition to a oneway ANOVA, multi-factorial ANOVA was also used to infer interactions between parameters. Calculations of basic statistical parameters (geometric, arithmetic means, etc.) and ANOVA analysis were performed with SPSS (Chicago, IL).
3. Results The recoveries achieved by the analytical method used in the study were within 715% of the certified reference values for all elements except As (Table 1). The recovery for As (145% higher than the value quoted in the SRM) was outside the acceptance (15%) criteria. It should be noted that arsenic in the reference material (0.05 mg/g) was not certified. The higher recovery of this element may be due to two factors: (a) the certified value may not be the true concentration, and (b) arsenic is a difficult element to analyze by ICP-MS and the disparity in values may reflect, at least partially, the uncertainty in the quantification of very low levels of As in the biological materials. The analytical detection limits for the six elements discussed in this paper were 0.28 mg/g for Zn, 0.08 mg/g for Cd, 0.04 mg/g for Cu and 0.01 mg/g for As, Se and Pb (Table 1).
Fig. 1. Map of Jamaica (not drawn to scale) showing the distribution of farm locations where samples used in the study were derived. The parishes are labeled as follows: 1, Hanover; 2, Westmoreland; 3, Saint James; 4, Saint Elizabeth; 5, Trelawny; 6, Manchester; 7, Saint Ann; 8, Clarendon; 9, Saint Catherine; 10, Saint Mary; 11, Saint Andrew; 12, Portland; 13, Saint Thomas.
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Table 1 Quality assurance data from the determination of As, Cd, Cu, Pb, Se and Zn in samples and the standard reference material (N ¼ 10) Material analyzed
Average metal concentrations (mg/g dry wt)
Standard reference material Sample batches Average % recovery Detection limit a
As
Cu
Zn
Se
Cd
Pb
0.05a 0.0770.02 145 0.01
16078.0 15379.6 95.8 0.037
127716 113717 89.1 0.28
0.7370.06 0.64 88.0 0.01
0.5070.03 0.4070.04 85.6 0.08
0.1370.01 0.1170.01 86.2 0.01
Value not certified.
Table 2 Arithmetic averages, geometric means and ranges in concentrations of Pb, Cd, As, Zn, Cu and Se in bovine kidney and liver samples in mg/g dry weight (wet weight in parentheses) Element
Liver (N ¼ 100) Averagea
Pb Cd As Zn Cu Se
GMa b
0.162 (0.052) 10.1 (3.24) 0.050 (0.016) 92.2 (29.5) 63.9 (20.4) 1.35 (0.432)
K/L ratioc
Kidney (N ¼ 100)
0.071 1.18 0.027 86.9 31.5 0.974
(0.023) (0.378) (0.009) (27.8) (10.1) (0.312)
Range
Averagea
GMa
Range
BDL–1.30 (BDL–0.416) BDL–256 (BDL–82.1) BDL–1.12 (BDL–0.358) 16.6–229 (5.28–73.6) BDL–312 (BDL–99.8) 0.180–8.48 (0.058–2.71)
0.523 (0.126) 33.1 (7.92) 0.106 (0.025) 84.8 (20.4) 16.2 (3.89) 4.23 (1.02)
0.292 (0.070) 6.17 (1.48) 0.056 (0.013) 79.3 (19.0) 14.0 (3.36) 3.44 (0.825)
0.017–3.57 (0.004–0.857) 0.052–487 (0.012–117) 0.004–1.36 (0.001–0.326) 13.2–188 (3.17–45.2) BDL–91.2 (BDL–21.9) BDL–14.9 (BDL–3.58)
4.1 5.2 2.1 0.91 0.45 3.5
Note: average blank value was subtracted from each individual sample value. a Average: arithmetic average; GM: geometric mean; BDL: below the instrumental detection limit shown in Table 1. b Values in parentheses are in wet weight. c K/L is the ratio of average concentration in the kidney to average concentration in liver samples.
The geometric means (GM), arithmetic averages and ranges in concentrations of Pb, Cd, As, Zn, Cu and Se in the kidney and liver samples both on wet-weight and dry-weight basis are shown in Table 2. Measured average water contents for the kidney and liver samples were 7675.5% and 6872.4%, respectively. To convert the concentrations given for dry-weight to wet-weight basis, the values should be multiplied by 0.24 for the kidney and 0.32 for liver samples to account for the water contents of these organs. We explored the influence of age and gender on the concentrations of the trace metals using multiple linear regression models. Age was only found to be a predictor of Cd (t ¼ 4.38; po0.001) in the kidney and was not significantly associated with any element in the liver (Table 3). The Cd contents of older cows were much higher than those of younger animals. Gender was a predictor of the Cu in kidneys of the test animals (t ¼ 3.01; p ¼ 0.004) with levels in female animals being higher than those of male cows. Female cows also had more Se in their livers than the males (t ¼ 2.39; p ¼ 0.019) (Table 3). Non-parametric Spearman correlation was performed to examine the inter-element relationships in cattle organs and the results are presented in Table 4. For liver samples, weak positive associations were found between copper and zinc (p ¼ 0.044, r ¼ 0.198), as well as zinc and lead (p ¼ 0.016, r ¼ 0.239); moderate positive associations was shown between cadmium and zinc (p ¼ 0.0002, r ¼ 0.361); while a moderate negative association between copper and arsenic (p ¼ 0.0009, r ¼ 0.327) was found (Table 4). The kidney samples showed a very strong positive correlation between cadmium and zinc (po0.0001, r ¼ 0.70); a positive correlation between copper and zinc (p ¼ 0.019, r ¼ 0.233), arsenic and selenium (p ¼ 0.013, r ¼ 0.247) and lead and selenium (p ¼ 0.007, r ¼ 0.269). The inter-organ relationships were compared for each metal (Table 4) and only weak positive associations were detected for cadmium (p ¼ 0.019, r ¼ 0.235) and selenium (p ¼ 0.015, r ¼ 0.243).
Table 3 Multiple linear regression of age and gender versus (log transformed) concentrations of trace metals in kidney and liver samples Cd Age (kidney) t 4.38 p-value o0.001
Cu
Pb
Zn
Se
As
1.30 0.197
1.26 0.211
1.07 0.287
0.799 0.427
0.571 0.570
1.46 0.149
1.35 0.182
0.890 0.377
1.15 0.256
0.256 0.799
0.605 0.547
Gender (kidney) t 0.493 p-value 0.624
3.01 0.004
0.146 0.884
0.911 0.365
1.39 0.116
1.53 0.131
1.58 0.117
0.463 0.645
0.301 0.764
2.39 0.019
0.088 0.930
Age (liver) t p-value
Gender (liver) t p-value
0.068 0.946
4. Discussion The ratio of average metal concentration in kidney to that in liver (K/L ratio) was highest for Cd (5.2) and was elevated for Pb (4.1), Se (3.5) and As (2.1) (estimated from data in Table 2). By contrast, Cu is depleted in the kidney relative to the liver (K/L ratio ¼ 0.45) and there does not appear to be a preferential accumulation of Zn in the kidney relative to the liver (K/L ratio ¼ 0.91). 4.1. Deficiency/toxicity of the trace metals The geometric mean concentration of Pb in the liver samples (0.07 mg/g wet weight (ww)) falls in the range of 0.05–0.40 mg/g ww that have been reported in many countries (Alonso et al., 2000). Our geometric mean value for Pb in the liver (0.02 mg/g ww), however, is less than the 0.05–0.46 mg/g found in previous
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Table 4 Spearman rank correlation coefficients for log metal concentrations in the animal tissues (N ¼ 100) Cadmium (Cd) Liver samples Cadmium (Cd)
Copper (Cu)
Zinc (Zn)
Arsenic (As)
Selenium (Se)
Lead (Pb)
r ¼ 0.127 p ¼ 0.208
r ¼ 0.361 p ¼ 0.0002
r ¼ 0.126 p ¼ 0.213
r ¼ 0.138 p ¼ 0.171
r ¼ 0.033 p ¼ 0.745
r ¼ 0.198 p ¼ 0.044
r ¼ 0.327 p ¼ 0.0009
r ¼ 0.131 p ¼ 0.197
r ¼ 0.079 p ¼ 0.435
r ¼ 0.030 p ¼ 0.766
r ¼ 0.031 p ¼ 0.759
r ¼ 0.239 p ¼ 0.016
r ¼ 0.115 p ¼ 0.257
r ¼ 0.56 p ¼ 0.582
Copper (Cu)
r ¼ 0.127 p ¼ 0.208
Zinc (Zn)
r ¼ 0.361 p ¼ 0.0002
r ¼ 0.198 p ¼ 0.044
Arsenic (As)
r ¼ 0.126 p ¼ 0.213
r ¼ 0.327 p ¼ 0.0009
r ¼ 0.030 p ¼ 0.766
Selenium (Se)
r ¼ 0.138 p ¼ 0.171
r ¼ 0.131 p ¼ 0.197
r ¼ 0.031 p ¼ 0.759
r ¼ 0.115 p ¼ 0.257
Lead (Pb)
r ¼ 0.033 p ¼ 0.745
r ¼ 0.079 p ¼ 0.435
r ¼ 0.239 p ¼ 0.016
r ¼ 0.560 p ¼ 0.582
r ¼ 0.609 p ¼ 0.549
r ¼ 0.024 p ¼ 0.804
r ¼ 0.700 po0.0001
r ¼ 0.047 p ¼ 0.645
r ¼ 0.023 p ¼ 0.819
r ¼ 0.155 p ¼ 0.122
r ¼ 0.233 p ¼ 0.019
r ¼ 0.141 p ¼ 0.163
r ¼ 0.079 p ¼ 0.436
r ¼ 0.006 p ¼ 0.950
r ¼ 0.019 p ¼ 0.854
r ¼ 0.111 p ¼ 0.273
r ¼ 0.154 p ¼ 0.125
r ¼ 0.247 p ¼ 0.013
r ¼ 0.137 p ¼ 0.176
Kidney samples Cadmium (Cd)
r ¼ 0.609 p ¼ 0.549
Copper (Cu)
r ¼ 0.024 p ¼ 0.804
Zinc (Zn)
r ¼ 0.700 po0.0001
r ¼ 0.233 p ¼ 0.019
Arsenic (As)
r ¼ 0.046 p ¼ 0.645
r ¼ 0.141 p ¼ 0.163
r ¼ 0.019 p ¼ 0.854
Selenium (Se)
r ¼ 0.023 p ¼ 0.819
r ¼ 0.079 p ¼ 0.436
r ¼ 0.111 p ¼ 0.273
r ¼ 0.247 p ¼ 0.013
Lead (Pb)
r ¼ 0.155 p ¼ 0.122
r ¼ 0.006 p ¼ 0.950
r ¼ 0.154 p ¼ 0.125
r ¼ 0.137 p ¼ 0.176
r ¼ 0.269 p ¼ 0.007
r ¼ 0.235 p ¼ 0.019
r ¼ 0.039 p ¼ 0.696
r ¼ 0.098 p ¼ 0.332
r ¼ 0.012 p ¼ 0.905
r ¼ 0.243 p ¼ 0.015
Inter-tissue relationships Kidney–liver metal correlation
r ¼ 0.269 p ¼ 0.007
r ¼ 0.037 p ¼ 0.715
po0.05. po0.01. po0.001.
studies in other countries (Alonso et al., 2000). The low average values are consistent with the fact that neither the liver nor the kidney is an accumulator organ for Pb in mammals (Kramer et al., 1983; Salisbury et al., 1991; Alonso et al., 2000). Concentrations of As in the bovine kidney (o0.015–0.07 mg/g) and liver (o0.015–0.05 mg/g) reported in the literature (Kramer et al., 1983; Vos et al., 1987; Jorhem et al., 1991; Salisbury et al., 1991) are somewhat higher than our geometric mean values of 0.013 mg/ g ww for kidney and 0.01 mg/g ww for liver. Our mean Cd concentrations in the liver (0.38 mg/g ww) and kidney (1.5 mg/g ww) are much higher than the 0.03–0.12 mg/g ww for livers and 0.07–0.65 mg/g ww for kidneys that have been reported in several countries (Nriagu, 1981; Alonso et al., 2000). The enrichment of Jamaican samples with Cd can be expected considering the elevated levels of this element that have been reported in local soils (Engel et al., 1996; Lalor, 1995). Any comparison of our data with those of other studies must be made in a circumspect manner because of differences in analytical methodologies used, physiological conditions of the cows (whether lactating, pregnant
or sickly), age classification of the animal population and the nature of the feed (Olsson et al., 2001; Wlostowski et al., 2006). Reports in the scientific literature (Kramer et al., 1983; Salisbury et al., 1991) typically show Se concentrations of 0.23–0.31 for bovine livers and 0.92–1.5 mg/g for bovine kidneys. These values are consistent with our geometric mean values of 0.82 mg/g ww for the kidney and 0.31 mg/g ww for liver samples (Table 2). According to the criteria developed by Puls (1994), Se would be considered to be deficient (o0.54 mg/g dw) in 28% of the liver samples from our study animals, marginally deficient (0.58–0.86 mg/g dw) in 15% of the samples, adequate to high (0.86–4.24 mg/g dw) in 50% and at chronic toxicity level (44.3 mg/g dw) in 5% of the liver samples. The geometric mean renal Cu concentration in Jamaican bovine samples (3.4 mg/g) is among the lowest reported for cattle in any part of the world (see Alonso et al., 2000; Miranda et al., 2005). Also, the geometric mean hepatic Cu concentration of 10 mg/g found in our study is well below average values of 23–137 mg/g ww that have been reported for cattle in other parts
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of the world (Alonso et al., 2000; Miranda et al., 2005). Two-thirds of the animals studied in this project showed hepatic Cu values below 25 mg/g considered to be normal for cattle (Puls, 1994). Copper concentrations in 48% of the liver samples were below 34 mg/g dry weight (dw) considered to be the deficiency threshold while 26% of the renal values were in the range of 34–85 mg/g dw considered to be the marginally deficient level (Puls, 1994). Interestingly, Cu levels in 52% of the renal samples were o12 mg/g ww found in livers of moose showing ‘‘moose wasting disease’’ associated with Cu deficiency in Alaska, Sweden and Nova Scotia (Frank et al., 2000; O’Hara et al., 2001; Custer et al., 2004; Gamberg et al., 2005). Liver Cu concentration is used to diagnose Cu deficiency in livestock (National Research Council, 1984) and our results for a large number of cows on the island would seem to be in the less than optimal range. Copper deficiency is associated with a number of physiological and biochemical changes including reduced appetite, growth and immune function impairment and increased susceptibility to disease (Underwood, 1971; Dorton et al., 2003). Copper deficiency also impairs normal metabolism, resulting in microcytic, hypochromic anemia and associated hepatic Fe overload, altered structure and functioning of circulating and immune cells, aberrant cardiac electrophysiology and persisitent effects on neurobehavior and the immune system (Uriu-Adams and Keen, 2005; Auclair et al., 2006). There is nothing to indicate that the cattle population in Jamaica is experiencing any of these adverse health effects associated with Cu deficiency, but then the health status of farm animals on the island is not well documented. Copper deficiency is now recognized to be a common problem in many domesticated and wild animals in many countries (UriuAdams and Keen, 2005; Blanco-Penedo et al., 2006), and Jamaica should be added to the list. Copper is commonly supplemented to most feedlot diets in the US and other countries (Dorton et al., 2003), in recognition of the fact free range foraging (a-la in Jamaica) does not provide cattle with adequate supply of this essential microelement. The geometric mean Zn concentration for kidneys (19 mg/g ww) falls within the range of 14–30 mg/g ww found in other studies and within the range considered adequate for cattle tissue (Puls, 1994). The mean value for livers (28 mg/g), however, is lower than 39–70 mg/g reported in many countries (Alonso et al., 2000; Miranda et al., 2005). About 15% of the hepatic samples had Zn concentrations below 20 mg/g and the question of Zn insufficiency in some of theses animals may be raised. Low liver Zn levels in cattle have been associated with exposure to high environmental Cd pollution (Miranda et al., 2005) and the low hepatic Zn concentrations in our samples may be related to high levels of Cd in local soils. About 70% of the variance in hepatic Cd concentrations in our samples (Table 4) may reflect the effects of Zn metabolism or other determinants of Zn concentration in the kidney. The levels of Cd in Jamaican soils may be related to low Cu concentrations in livestock tissues since other studies have reported hypocupraemia in areas where soils are polluted with Cd (Koh and Judson, 1986; Spierenburg et al., 1988; Wentink et al., 1988; Prankel et al., 2005). Whereas deficiencies in essential micronutrients (Cu and Zn) may be an issue, the bioaccumulations of non-essential metals in kidneys and livers may reach levels of concern toxicologically (Nriagu, 1986). For mammals, the threshold levels of biological effects for Cd have been reported to be 20–200 mg/g in the liver and 50–400 mg/g in the kidney (Fisk et al., 2005). Only two liver and two kidney samples from our test animals fell into these ranges. The kidney, liver, lung, heart and testis are the main target organs following Cd exposure and the residence times of Cd in these animal tissues are years-to-decades long (Saratug et al.,
2003). Because of the long retention times and the fact that Cd is not physiologically regulated in mammals, sustained environmental exposures often result in significant accumulation of Cd in these organs in a time-dependent manner. Many previous studies, like ours, have found significant associations between renal and hepatic Cd concentrations with age for a wide variety of organisms including humans (Komarnicki, 2000; Olsson et al., 2001; Saratug et al., 2003; Gamberg et al., 2005).
4.2. Inter-element relationships Because of the role of the liver and kidney in the metabolism of trace metals and the susceptibility of these organs to metal accumulation and toxicity (Underwood, 1971; Taylor, 1996; Alonso et al., 2004), the inter-element relationships in these tissues are generally of concern and scientific interest. Significant interelement relationships provide some indication of the moderating effects on observed tissue accumulations of the metals. The strongest positive interaction observed in our samples was between Cd and Zn in the kidney (po0.0001, r ¼ 0.700) and liver (p ¼ 0.0002, r ¼ 0.361) (Table 4). Statistically significant associations have been likewise reported previously between Zn and Cd (as well as Cu, Pb and other trace metals) in various mammalian tissues (Bebe and Panemangalore, 1996; Taylor, 1996; Frank et al., 2000; Komarnicki, 2000; Coudray et al., 2006; Phillips et al., 2005; Reeves and Chaney, 2004). The liver and a number of tissues contain an ubiquitous Zn binding protein, metallothioneins (MTs), which have at least two function—to sequester Zn and release it by events that signal its requirement (Tapiero and Tew, 2003). These proteins also bind Cd exquisitely. Besides a role in metal homeostasis, the MTs can also serve as store for Zn and Cd, and be involved in metal transfer (Sharma et al., 2005; Phillips et al., 2005). The mechanisms by which Cd affects Zn metabolism are not yet well understood but the strong association of Cd and Zn in the liver and kidney most likely is related to the fact that the handling of Cd2+ and Zn2+ in these tissues intersect at several points. Subchronic dietary exposure to Cd has been linked with collateral increases in Zn and Cu concentrations in the liver and kidney (Phillips et al., 2005). It is conceivable that the significant correlation between Zn and Cu concentrations in the liver (p ¼ 0.044, r ¼ 0.198) and kidney (p ¼ 0.019, r ¼ 0.233) observed in our samples may stem from the indirect effect of co-exposure to high levels of Cd in local soils. The associations may also be related to the sharing of transport systems by the metals and the regulatory effects of MTs which can be independently expressed by any of these three metals. Besides the bones which are the main target organ for Pb accumulation in vertebrates, chronic exposure often results in elevated levels of this metal in the kidney compared to the liver (Ma, 1989; Komarnicki, 2000). The fact that the liver is not accumulator sites for Pb is suggested by the low hepatic Pb level (0.07 mg/g) and high K/L ratio (of 44) found in our samples (Table 2). We found no significant association between the amounts of Pb in the kidney and liver (Table 4), suggesting that the lead pools in these tissues may be unrelated to each other. A number of studies have reported positive correlations between hepatic Zn and Pb concentrations at relative low levels of exposure (Alonso et al., 2002; Rahil-Khazen et al., 2002) while others have found no association (Szefer et al., 1994; Alonso et al., 2004). Although other studies have also reported significant associations between Pb and Zn, Pb and Cu, and Pb and Cd in the kidney (August et al., 1989; Alonso et al., 2002, 2004; RahilKhazen et al., 2002; Miranda et al., 2005), we did not find such correlations in Jamaican samples (Table 4).
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At high doses of exposure, As is said to have a detoxifying effect against Se and vice versa (Bedwal et al., 1993). At low levels of exposure, cells show less selectivity towards selenate and arsenate ions so that a deficiency in one often engenders increased uptake of the other in the liver and kidney (Nriagu, 1994; Miyazaki et al., 2005). The significant positive association between the renal As and Se concentrations (p ¼ 0.013, r ¼ 0.247) in our samples is consistent with sympathetic accumulation of As and Se when the pools of these elements are low in the liver.
4.3. Possible health risks of trace metal levels found in kidney and liver Kidney and liver represent a significant vector for trace metal exposure in some segments of the population of Jamaica (Lalor et al., 2004). Consumption of kidney and liver also represents a significant source of vitamins, proteins, lipids, antioxidants, essential micronutrients and calorific intake for the island population. The nutritional benefits can be substantial and must therefore be weighed against the potential health risks associated with toxic metals that have accumulated in these organs. The goal of this section is not to provide a detailed analysis of the weight of evidence and scientific information on risk and benefits of offal consumption. Since data required for comprehensive risk assessment is lacking, the aim of this section rather is to draw attention and encourage further investigation of potential health effects associated with exposure to As, Cd, Cu, Pb, Se and Zn found in kidneys and livers of locally raised cows. A number of countries have set maximum residue levels (MRL) for these organs to protect human health. There are currently no such guidelines for metal concentrations in animal meats and organs sold in Jamaica and the values for a number of countries
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are listed in Table 5. Whether the guidelines for other countries are valid for Jamaican population is a matter for conjecture at this time. The foreign guidelines are used here merely as a convenient benchmark for discussing our data. With the exception of Cd, the geometric mean concentrations of trace elements measured in liver and kidney of Jamaican cattle are below the MRLs that have been set in many countries (Table 5). When the distributions of individual elements are considered, About 60% of the kidney samples showed Cd values above 1.0 mg/g while 40% of the liver samples had Cd concentrations above 0.5 mg/g—MRL in many countries (Table 5). No sample had Cu, Pb, Zn or As content that was above the recommended guidelines for either the kidney or liver. It should be emphasized that the MRLs in themselves cannot guarantee low-risk exposures (they do not address eating habits, smoking, environmental sources and nutritional factors; Berti et al., 1998) and are used as an expedient tool because they are measurable and enforceable, in principle. Dietary data were recently collected from 723 adults in the central part of the island using food frequency questionnaire (Nriagu, in preparation). Average consumption rates for kidney and liver calculated from this survey were 2.2 and 5.2 g/day, respectively. From current data on hepatic and renal metal concentrations, the average intake of each metal from consumption of the two tissues has been estimated and shown in Table 6. The mean intake of Cd from consumption of both kidney and liver is estimated to be 5.2 mg/day, equivalent to about 7% of the provisional tolerable daily intake (PTDI) value of 72 mg/day recommended by WHO (1989). It should, however, be noted that about 5% of the liver samples analyzed had over 40 mg/g Cd while 15% of the kidney samples had over 40 mg/g Cd. Anyone who habitually consumes kidneys or livers with such elevated levels of Cd may be at some risk of exceeding the PTDI. From our
Table 5 Maximum residue limits (mg/g wet weight) for arsenic, cadmium, lead, copper, zinc and selenium for bovine liver and kidney in various countries and organizations Liver
Kidney
Country
Reference
Lead
0.5 0.5 1 0.6 2
0.5 0.5 1 1.0 1
World Health Organization European Commission Slovac Republic Russia United Kingdom
WHO (2001) EU Commission (1997, 2001) Kottferova and Korenekova (1995) Farmer and Farmer (2000) UK Minisitry of Agriculture, Fisheries and Food (1998)
Cadmium
0.5–1 0.5 0.5 1 0.1
1 0.5 1 1 0.3
European Commission Germany Slovac Republic Australia Russia
EU Commission (1997, 2001) Kreuzer et al. (1988) Kottferova and Korenekova (1995) Kramer et al. (1983) Farmer and Farmer (2000)
Arsenic
1 2 2
1 2 2
Australia Canada World Health Organization
Kramer et al. (1983) Salisbury et al. (1991) WHO (1992)
Copper
100 100 150 60
100 100 150 60
United States Australia Canada Slovac Republic
NAS (2000) Langlands et al. (1988) Salisbury et al. (1991) Kottferova and Korenekova (1995)
Zinc
500 500 150 100 80 100
500 500 150 100 80 100
United States Canada Australia Canada Slovac Republic Russia
NAS (2000) National Research Council (1984) Langlands et al. (1988) Salisbury et al. (1991) Kottferova and Korenekova (1995) Farmer and Farmer (2000)
2 2
2 2
United States World Health Organization
NAS (2000) WHO (1989)
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Table 6 Estimated exposure to trace metals from consumption of kidney and liver compared to the provisional tolerable daily intake (PTDI) for toxic metals or recommended dietary allowance (RDA) for the essential trace elements Element
Average daily consumption (mg/day)
Established guideline
Kidney
Liver
Kidney+liver
Arsenic Cadmium Copper
0.03 3.26 7.39
0.45 1.96 52.4
0.48 5.22 59.79
Lead Selenium Zinc
0.154 1.82 41.9
0.118 1.62 145
0.272 3.44 186.9
results, it would appear that kidneys and livers of a small number of cattle reared in Jamaica may be a health risk to some consumers. It must be emphasized that many people on the island do not eat either kidney or liver and that the risk of exposure to Cd in offal depends on dietary habits, nutritional factors, and presence of diabetic/kidney diseases, smoking, consumption of other types of foods that contain elevated levels of Cd, gender and genetic diseases (Jarup et al., 1998; Meeus et al., 2002). The mean intakes of Pb (0.27 mg/day), As (0.48 mg/day) and Se (3.4 mg/day) from kidney and liver (Table 6) make up o1%, o1% and 7% of the PTDI, respectively; offal represents an insignificant route of exposure for these elements. In terms of the essential trace metals, an average daily consumption of kidney and liver yields 0.98 mg Cu, well below the recommended daily allowance of 9 mg/day in the United States (Johnson et al., 1992; Uriu-Adams and Keen, 2005). The daily intake of Zn from kidney and liver is 0.18 mg, also well below the RDA value of 8 mg/day for women and 11 mg/day for men in the USA (IOM, 2001). The consumption of offal from local animals thus does not appear to be an important dietary source of either Cu or Zn for people on the island.
5. Conclusions The accumulation of trace metals in kidneys and livers of cows in Jamaica is enigmatic. On the one hand, the data suggest that many cattle may be experiencing Cu and, to a lesser extent, Zn deficiency. One consequence of the low Cu and Zn contents is that consumption of offal from local cows does not represent a significant dietary source of the two essential microelements to the local population. On the other hand, Cd levels are elevated in these organs and consumption of large quantities of the offal represents a potential health hazard. There is clearly a need for additional studies to assess the concatenated effects of exposing local cattle to suboptimal amounts of Zn and Cu and elevated doses of Cd.
Acknowledgments Sample collection and preparation were done while the lead author held a senior Fulbright Fellowship at the International Center for Environmental & Nuclear Sciences, University of the West Indies, and financial support for the work is acknowledged. Appreciation is extended to Ms. Myriam Afeiche for assistance with data analysis. Human subjects: The research did not involve any human subjects, hence there was no need for institutional review board (IRB) approval.
PTDI ¼ 154 mg/day (WHO, 2002) PTDI ¼ 72 mg/day (WHO, 2002) RDA ¼ 0.9 mg/day (IOM, 2001) PTDI ¼ 10 mg/day (IOM, 2002) PTDI ¼ 257 mg/day (WHO, 2002) RDA ¼ 55 mg/day (NAS, 2000; ATSDR, 2003) RDA ¼ 8 mg/day for women and 11 mg/day for men (IOM, 2000)
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