Associated factors for higher lead and cadmium blood levels, and reference values derived from general population of São Paulo, Brazil

Science of the Total Environment 543 (2016) 628–635

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Associated factors for higher lead and cadmium blood levels, and reference values derived from general population of São Paulo, Brazil Carmen Silvia Kira a,⁎, Alice Momoyo Sakuma a, Eduardo Mello De Capitani b, Clarice Umbelino de Freitas c, Maria Regina Alves Cardoso d, Nelson Gouveia e a

Instituto Adolfo Lutz, Centro de Materiais de Referência, Av. Dr. Arnaldo, 355, São Paulo, SP CEP 01246-000, Brazil Universidade Estadual de Campinas — UNICAMP, Faculdade de Ciências Médicas, Departamento de Clínica Médica, Centro de Controle de Intoxicações, Brazil Secretaria de Estado da Saúde/SP, Coordenadoria de Controle de Doenças, Brazil d Universidade de São Paulo, Faculdade de Saúde Pública, Departamento de Epidemiologia, Brazil e Universidade de São Paulo — USP, Faculdade de Medicina, Departamento de Medicina Preventiva, Brazil b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The exposure of population of São Paulo city to lead and cadmium is low. • Pb level was associated with gender, smoking, offal intake, area of residence, age. • Cd level was associated with gender, smoking, distilled beverages, age. • RV for Pb in blood for children and adolescents were 29 and 31 μg/L, respectively. • RV for Cd in blood for children and adolescents were 0.2 and 0.6 μg/L, respectively.

a r t i c l e

i n f o

Article history: Received 4 August 2015 Received in revised form 12 November 2015 Accepted 13 November 2015 Available online xxxx Editor: D. Barcelo Keywords: Reference values Lead Cadmium Blood

⁎ Corresponding author. E-mail address: [email protected] (C.S. Kira).

http://dx.doi.org/10.1016/j.scitotenv.2015.11.067 0048-9697/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t Human activities are associated with emissions of various metals into the environment, among which the heavy metals lead and cadmium stand out, as they pose a risk to human life even at low concentrations. Thus, accurate knowledge of the levels of these metals exhibited by the overall population, including children, is important. The aim of this study was to estimate the concentrations of lead and cadmium in the blood of adults, adolescents and children residing in the city of São Paulo, assess factors associated with higher lead and cadmium blood levels, and to establish reference values for this population. The study sample consisted of 669 adults over 20 years old, 264 adolescents aged 12 to 19 years old and 391 children under 11 years old from both genders. The samples were collected at the end of 2007 and during 2008 in different city zones. Higher blood lead concentration was significantly associated with gender, smoking, offal intake, area of residence and age. The blood cadmium concentration was significantly associated with gender, smoking, consumption of distilled beverages and age. The reference values of lead and cadmium established for adults above 20 years old were 33 μg/L and 0.6 μg/L, respectively, for adolescents (12 to 19 years old) were 31 μg/L and 0.6 μg/L, respectively and for children under 11 years old were 29 μg/L and 0.2 μg/L, respectively.

C.S. Kira et al. / Science of the Total Environment 543 (2016) 628–635 Environmental monitoring Environmental health

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The results of this study indicate that the exposure levels of the investigated population to lead and cadmium are low. © 2016 Elsevier B.V. All rights reserved.

1. Introduction High levels of urbanization and the expansion of industry have contributed to the contamination of environmental compartments by heavy metals. Large amounts of waste accumulate in the environment, affecting human health, making these waste products a cause of much concern (Valavanidis and Vlachogianni, 2010). Among the heavy metals, lead and cadmium are considered to be widely distributed environmental pollutants. Both are characteristically persistent because they do not degrade in nature; thus, once they are released into the environment, they remain in circulation over long periods of time (Bernard, 2008; Mehmood et al., 2014; Nordic Council of Ministers, 2003). Human exposure to lead resulting from the contribution of natural factors is low. However, the extensive use of this metal in various sectors has resulted in a significant increase of its concentration in the environment (ATSDR, 2007; WHO, 1995). Mining, smelting, waste incineration, acid-battery recycling, coal and oil combustion, and processes in the plastic, ceramic and rubber industries are anthropogenic sources of lead emissions into the environment (Karrari et al., 2012; Minozzo et al., 2008). The use of lead as a gasoline additive has also been, in some countries, an important source accounting for the increase of the concentration of this element in the environment (ATSDR, 2007). The main route of human exposure to cadmium is the diet, while cigarettes are a relevant source of exposure for smokers (Chasin and Cardoso, 2003; Madeddu et al., 2011; Nordberg et al., 2007; Nordic Council of Ministers, 2003). Lead and cadmium are toxic elements because they can cause adverse effects on human health, even at low levels of exposure (Pourmand et al., 2012). Lead can affect the central nervous, cardiovascular and the hematological system (ATSDR, 2007). The main toxicity of cadmium is associated with renal dysfunction, as it accumulates in the kidneys (Järup et al., 1998; Nordic Council of Ministers, 2003). Although lead and cadmium emissions into the environment have decreased dramatically in recent years, these metals still pose a problem to the overall population because they tend to bioaccumulate along the food chain, eventually reaching humans (Chasin and Cardoso, 2003; Nordic Council of Ministers, 2003; WHO, 1992). Children and adolescents represent a special group within the overall population because, as a function of the particular characteristics of their bodily development, they are more susceptible to environmental pollutants and are consequently more vulnerable. Therefore, children and adolescents should be considered in biomonitoring studies. Reference values indicate the baseline exposure of a population to a given substance during a given period of time and may be used to identify increased exposure in an individual and in a collective basis (Ewers et al., 1999). Reference values are usually defined as the 95th percentile or upper confidence interval of the 95th percentile of the concentration values (Ewers et al., 1999; Wilhelm et al., 2004). Population-based reference values have not yet been established in Brazil, but some specific studies sought to establish regional reference values for blood lead (Kuno et al., 2013; Paoliello et al., 2001) and cadmium (Kuno et al., 2013) in adults. Despite the importance of biomonitoring, this type of study has not yet been regularly performed in Brazil. Thus, the aim of the present study was to estimate the concentrations of lead and cadmium in the blood of adults, adolescents and children residing in the city of São Paulo, assess factors associated with higher lead and cadmium blood

levels, and to establish reference values (RV) for these metals in the study population. 2. Materials and methods 2.1. Study design In this cross-sectional study, lead and cadmium were measured in the blood of “healthy” children, adolescents and adults who reside in the city of São Paulo. The samples were collected at the end of 2007 and during 2008 in five different city zones (North, South, East, West and Central). Initially the study was design to collect biological samples of adults from 14 to 70 years old, and from children aged 6 to 13 years old living in São Paulo city. Aiming to derive reference values the study population was stratified in three subgroups in order to comply with three different groups, that is, children, adolescents, and adults. Although the International Federation of Clinical Chemistry (IFCC) recommends including a minimum of 120 observations to estimate reliable reference values (Solberg, 1987) in this study, we estimated a sample size of 500 children and 500 adults. The reason for this design was that the aim of the present study was to establish reference values of various metals, the variations of which were not previously known in the blood levels in the non-exposed population. In addition, the various age ranges would still be stratified according to several variables. The study sample was probabilistic, and the participants were selected by means of two-stage cluster sampling. In stage one, primary sampling units (PSUs) were selected, corresponding to census areas established by the Brazilian Institute of Geography and Statistics (Instituto Brasileiro de Geografia e Estatística — IBGE). In stage two, secondary sampling units (SSUs) were selected, corresponding to the households in each PSU selected in the first stage. 2.2. Inclusion and exclusion criteria To establish reference intervals of lead and cadmium in the blood of healthy individuals, the following criteria were established for the selection of participants: Inclusion criteria: “healthy” school children (6 to 13 years old) and “healthy” adults, aged 14 to 70 years old, of both genders and residing for at least one year in the city of São Paulo. Exclusion criteria: history of drug use; chronic use of medication; diabetes mellitus; chronic alcoholism; arterial hypertension; liver, kidney, urinary or endocrine system diseases; past or present history of occupational exposure to the investigated metals; non-professional or artisanal activity involving manipulation of the investigated metals; residing in areas known as geologically or anthropogenically contaminated with any of the investigated metals; residing in São Paulo for less than one year; dental amalgam placed less than five days earlier; infection with human immunodeficiency virus (HIV); or presence of diseases requiring continuous medication. These conditions were identified through an eligibility questionnaire. 2.3. Data collection A preliminary interview was conducted using an eligibility questionnaire to select individuals without risk of exposure to excessive concentrations of metals. A second questionnaire was used to collect information on name, date of birth, gender, ethnicity, length of residence in São Paulo, address,

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occupation, smoking status, use of alcohol, use of medication or multivitamin supplements in the past 30 days, preferred diet (beef, chicken, fish, seafood) and socioeconomic status. Questionnaire data were collected by trained interviewers personally interviewing each participant or legal responsible person for the children included. No self-reported data were used in the study. 2.4. Blood collection Blood samples were collected by trained staff by means of venipuncture in the forearm using needles and vacuum tubes adequate for trace elements. Trace-element sodium heparin blue-cap 6-mL Vacuette tubes were used for sample collection. The samples were refrigerated in Styrofoam boxes containing recyclable ice for transport to the laboratory, which occurred on the same day as collection, including weekends. The metals were measured using an inductively coupled plasma mass spectrometer (ICP-MS, PerkinElmer, model Elan DRC II) equipped with an auto-sampler (PerkinElmer, model AS-93 plus). The standards were prepared in an ISO (International Organization for Standardization) class 7 clean room environment with ISO class 5 laminar flow; readings were performed in an ISO class 7 clean room environment to minimize the risk of contamination. Sample preparation consisted of a simple dilution (twenty-fold) with 0.05% Triton X-100 in 0.2% HNO3. The analytical method used was validated and performed according to Kira et al. (2014). The limits of quantification (LOQ) of lead and cadmium were 0.99 and 0.11 μg/L, respectively. The coefficients of variation (CV%) obtained on the same day and between days were 1.9% and 3.0% for lead and 2.0% and 5.4% for cadmium. The working range linearity was assessed, and the values of the resulting correlation coefficients were 0.9998 and 0.9996 for lead and cadmium, respectively. The analysis quality was assessed using Lyphocheck ® Whole Blood Metals Control Level 2 (Bio-Rad, Irvine, USA) as reference material; the accuracy for lead and cadmium were 89% and 87%, respectively. The study was approved by the Research Ethics Committee of the University of São Paulo School of Medicine (research protocol no. 260/ 10). Participation was voluntary; the participants were informed verbally and in writing of the study aims, the risks associated with the procedures for collection and the confidentiality of the results and signed an informed consent form. 3. Theory/calculation 3.1. Data analysis Statistical analysis was performed using the software SAS (SAS Institute Inc., 2011). Values below the LOQ were considered as ½ LOQ, as is often employed in biomonitoring studies (Becker et al., 2002; Link et al., 2007); extreme values were not excluded. In that way, the derivation of RV for lead and cadmium involved 1324 numerical results. The measured lead and cadmium concentrations were described as the geometric means; 5th, 10th, 50th, 90th and 95th percentiles; and 95% confidence intervals (CI) of the 95th percentiles. The distribution of the variables was investigated by means of the Kolmogorov–Smirnov (K–S) test. To identify the factors (gender; ethnicity; smoking; occupation; income; age; area of residence; educational level; frequency of offal, fish, seafood, meat and chicken intake; frequency of distilled beverage, wine and beer consumption) that could possibly exhibit association with the blood metal concentration, the variables were first subjected to bivariate analysis by means of the Mann–Whitney or the Kruskal– Wallis test. All the variables found to influence (p ≤ 0.20) the blood metal concentration on bivariate analysis were analyzed by means of a generalized linear model (GLM); only the statistically significant variables (p ≤ 0.05) were kept in the model.

The rounded values of the upper limits of the 95% CIs of the 95th percentiles of the distributions of the measured concentrations of both metals were used to derive the reference values. 4. Results Among the total number of households visited, 578 children met the eligibility criteria, but the parents of 39 of them refused authorization for blood collection, and one blood sample coagulated and could not be analyzed. Therefore, the final sample consisted of 538 children. Among the adults interviewed, 826 met the eligibility criteria, but 37 refused to provide blood samples, and three samples coagulated and could not be analyzed. Thus, 786 valid blood samples were included for analysis. Therefore, the final study sample consisted of 1324 individuals of both genders, including 669 adults (over 20 years old), 264 adolescents (12 to 19 years old) and 391 children (under 11 years old), who resided in the Central, Northern, Southern, Eastern and Western zones of São Paulo city. The general characteristics of the study population are shown in Table 1. The elements analyzed exhibited asymmetric distribution, even after several attempts at transformation. Thus, the data were analyzed by means of non-parametric statistics to derive the reference values. The percentile distributions of the blood lead and cadmium concentrations relative to various variables (age, gender, smoking and use of alcohol) and the suggested reference values are described in Tables 2 and 3, respectively. The reference values for lead in blood established in the present study for children (under 11 years old), adolescents (12 to 19 years old) and adults (over 20 years old) were 29 μg/L, 31 μg/L and 33 μg/L respectively. The reference values for cadmium in blood for children under 11 years old, adolescents (12 to 19 years old) and individuals over 20 years old were 0.2 μg/L, 0.6 μg/L, and 0.6 μg/L and 0.5 μg/L for men and women, respectively. Reference values and geometric means combined by sex and age were shown in Table 4. Table 5 describes the mean, mode, LOD, LOQ, minimum and maximum lead and cadmium concentrations in the blood of the investigated population, in addition to the number of samples with values below the LOQ of the method. Tables 6 and 7 present the results of multivariate analysis relative to lead and cadmium, respectively. The fitted model showed that the following variables were associated with the blood lead concentration: area of residence, gender, smoking, and offal intake. Relative to the area of residence, the fitted Table 1 Characteristics of the population, São Paulo, 2007–2008. Variable

Gender Male

Age 11 years old or less 12 to 19 years old 20 years old or more Education level Illiterate Fundamental High School University Family income ($ Reais)a Up to 1 mwb 1 to 3 mw 3 to 5 mw 5 to 10 mw More than 10 mw a b

Female

Total

n

%

n

%

n

%

207 123 263

34.9 20.7 44.4

184 141 406

25.2 19.3 55.5

391 264 669

29.5 19.9 50.5

3 299 87 33

0.7 70.9 20.6 7.8

4 361 189 49

0.7 59.9 31.3 8.1

7 660 276 82

0.7 64.4 26.9 8.0

54 333 80 53 22

10.0 61.4 14.8 9.8 4.1

80 400 105 59 18

12.1 60.4 15.9 8.9 2.7

134 733 185 112 40

11.1 60.9 15.4 9.3 3.3

$ Reais: Brazilian currency (1 USD = 2.15 Reais). mw: minimum wage (R$ 380 refers to the minimum wage in 2007).

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Table 2 Blood lead levels in relation to different variables and proposed reference intervals (μg/L), São Paulo, 2007–2008. Groups Age of subjects b11 12–19 N20 Gender Male Female Smoking habit Smokers Non-smokers Ex-smokers Drinking habita N1×/week b3×/month

N

P5

P10

P50

P90

P95

95% CI (P95)

RV

GM

393 264 667

11.2 11.5 12.8

13.1 13.9 14.4

16.8 18.0 19.4

23.1 22.9 24.0

26.7 26.6 30.4

25.0–28.9 24.5–30.5 27.8–33.0

29 31 33

17.1 17.7 19.1

593 731

13.1 11.4

15.2 13.2

19.0 18.2

26.7 21.6

30.5 24.5

28.9–32.8 23.2–27.8

33 28

19.1 17.6

161 1023 137

20.7 16.8 20.2

20.8 13.4 14.4

22.7 17.7 19.2

36.9 20.6 20.6

44.2 23.3 20.8

37.6–49.7 22.4–24.8 20.6–26.6

50 25 27

25.6 17.3 18.3

39 1285

15.1 11.7

17.4 13.8

20.9 18.4

32.3 22.9

33.5 27.8

31.7–46.9 26.2–29.4

47 29

22.3 18.1

CI: confidence interval; RV: reference value; GM: geometric mean. a Habit of drinking distillates.

model indicated statistically significant variation in blood lead concentration among Southern and Central zones of São Paulo. Significant differences in the blood lead concentration were also detected between the groups with different frequencies of offal intake and different smoking status and between males and females. The following variables were associated with the blood cadmium concentration in the fitted model: age range, gender, smoking and distilled beverages intake (Table 7). Spearman's correlation test was used to investigate the associations between lead and cadmium concentrations and age. The following results were obtained: age exhibited significant positive correlations with both the cadmium (r = 0.550, p b 0.001) and lead (r = 0.273, p b 0.001) concentrations.

5. Discussion In this study, reference values were established for blood lead and cadmium concentrations, stratified according to the variables age, gender, smoking and alcohol use for the adult population and for children. Regarding definition criteria for reference values (RV) the literature is quite inconsistent, each study defining it differently. Lately, the most used definition refers to the upper limit of the 95% CI of the 95th percentile of the distribution of values. Comparison between studies might not be so valid when means, medians or geometric means are used as reference values. Therefore, aiming to compare with biomonitoring studies we used the above definition for RV and the obtained values for the 95th percentile of the selected studies.

Few studies sought to establish reference values for the overall Brazilian population (Kuno et al., 2013; Paoliello et al., 2001). No study conducted in Brazil sought to derive reference values of lead or cadmium in children, and the number of such studies performed in other countries is small. Reference values for children were derived in the Czech Republic and Germany only (Cerná et al., 2012; Schulz et al., 2009). Germany and Czech Republic use as reference value the upper limit of the 95% CI of the 95th percentile of the distribution of values (Angerer et al., 2011; Batariová et al., 2006). One Brazilian study conducted in the city of Londrina from 1994 to 1996 reported reference ranges of 12–140 μg/L and 12–135 μg/L for men and women, respectively (Paoliello et al., 2001). The upper limits of the reference ranges established in Paoliello et al. (2001) are considerably higher than the ones derived in the present study: the differences are up to three times higher in the case of men over 20 years old and four times higher relative to women of the same age. The use of ethanol as a gasoline additive was made obligatory in Brazil since 1993, with a gradual phasing out process starting after this date (Paoliello and Chasin, 2001). This fact may account for the high levels of lead found among the population of Londrina, which still reflected the emissions of lead from gasoline into the environment. The reference levels for lead in blood corresponding to men and women obtained in a study conducted in the São Paulo metropolitan area with blood donors recruited in 2006 (Kuno et al., 2013) were higher compared to the ones derived in the present study (men: 60 μg/L versus 33 μg/L; women: 47 μg/L versus 28 μg/L). In addition to the city of São Paulo, the metropolitan area of São Paulo also includes other cities, such as São Bernardo do Campo, Guarulhos and Santo

Table 3 Blood cadmium levels in relation to different variables and proposed reference intervals (μg/L), São Paulo, 2007–2008. Groups Age of subjects b11 12–19 N20 Gender Male Female Smoking habit Smokers Non-smokers Ex-smokers Drinking habita N1×/week b3×/month

N

P5

P10

P50

P90

P95

95% CI (P95)

RV

GM

393 264 667

0.06 0.06 0.06

0.06 0.06 0.06

0.06 0.19 0.21

0.11 0.40 0.45

0.18 0.48 0.55

0.13–0.22 0.45–0.62 0.50–0.58

0.2 0.6 0.6

0.07 0.17 0.19

593 731

0.06 0.06

0.06 0.06

0.17 0.13

0.41 0.37

0.50 0.47

0.46–0.57 0.44–0.51

0.6 0.5

0.15 0.13

161 1023 137

0.24 0.06 0.15

0.27 0.06 0.19

0.44 0.06 0.27

0.62 0.25 0.38

0.68 0.32 0.45

0.64–0.79 0.28–0.35 0.39–0.52

0.8 0.4 0.5

0.42 0.11 0.27

39 1285

0.06 0.06

0.06 0.06

0.14 0.32

0.38 0.56

0.48 0.61

0.45–0.50 0.55–0.89

0.5 0.9

0.26 0.14

CI: confidence interval; RV: reference value; GM: geometric mean. a Habit of drinking distillates.

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Table 4 Percentiles, geometric means and reference values derived for cadmium and lead in blood, according to gender and age (in μg/L), São Paulo, 2007–2008.

Cadmium

Gender

Age

N

P5

P10

P50

P90

P95

95% CI (P95)

RV

GM

Male

b11 12 to 19 N20 b11 12 to 19 N20 b11 12 to 19 N20 b11 12 to 19 N20

209 123 260 184 141 406 209 123 260 184 141 406

0.06 0.06 0.06 0.06 0.06 0.06 11.6 13.9 15.1 10.1 11.4 12.1

0.06 0.1 0.06 0.06 0.06 0.06 13.3 14.8 16.6 12.0 11.9 13.5

0.06 0.3 0.2 0.06 0.1 0.2 17.0 18.5 19.8 16.5 17.4 19.2

0.1 0.5 0.5 0.1 0.3 0.5 25.4 26.4 27.5 21.7 20.0 22.1

0.2 0.6 0.6 0.1 0.4 0.5 29.4 29.4 33.1 23.2 22.9 27.8

0.2–0.4 0.5–0.7 0.5–0.6 0.1–0.2 0.3–0.5 0.5–0.6 25.8–32.6 26.6–32.0 29.5–42.3 21.8–26.5 20.3–24.5 24.1–31.1

0.4 0.7 0.6 0.2 0.5 0.6 32.6 32.0 42.3 26.2 24.5 31.1

0.07 0.3 0.2 0.07 0.1 0.2 17.9 18.7 20.4 16.3 16.8 18.4

Female

Lead

Male

Female

André, which are the seats of large industrial complexes, a fact that may have contributed to the higher blood lead levels (BLL). The reference values of lead in blood derived for men, women and children in the present study are lower compared to the ones established for the population of the Czech Republic. In that country, the reference values were derived based on data provided by a biomonitoring study performed from 2005 to 2009 (Cerná et al., 2012). In the Czech Republic, the use of leaded gasoline was restricted in 2001, which accounted for the high BLL found (Cerná et al., 2012). The reference values of BLL for adult men and women residing in the city of São Paulo were also lower compared to the ones established in Germany for this same sub-population group. In that country, the reference values for adults were set based on data corresponding to the period from 1997 to 1999; thus, there is a ten-year difference relative to the present study, reflecting levels of exposure over quite different time periods. The reference values established in older studies may be higher compared to the current exposure levels; therefore, their use may not be appropriate to analyze more recent biomonitoring studies. The reference value set for German children aged 3 to 14 years old was 35 μg/L (Schulz et al., 2009), based on data collected from 2003 to 2006. That value is slightly higher compared to the reference value derived in the present study for children under 11 years old (29 μg/L). Comparison of the reference values of BLL derived in the present study for adults (male and female) over 20 years old (42.3 μg/L and 31.1 μg/L, respectively) (Table 4) with the 95th percentiles reported in the population-based National Health and Nutrition Examination Survey (NHANES) (USA), namely, 44.1 μg/L and 30.0 μg/L for men and women, respectively, based on data collected in 2007/2008 (CDC, 2013), indicates that the values are quite similar. Relative to children under 11 years old, the reference values derived for São Paulo city, Table 5 Lead and cadmium blood concentrations of the study group (in μg/L), São Paulo, 2007– 2008. Lead

Cadmium

1324

1324

6.9

0.06

58.7

0.89

0

515

0.99

0.11

0.30

0.03

18.85

0.19

16.80

0.06

29 μg/L (Table 2), was slightly higher compared to the 95th percentile in the NHANES, 25.0 μg/L (CDC, 2013). Reference values derived in this study for adolescents aged 12 to 19 years old was 31 μg/L (Table 2), higher than the 95th percentile reported in the NHANES study of 19 μg/L (CDC, 2013), although rather similar with the reference values of 29.4 μg/L from adolescents of Italy (Pino et al., 2012). NHANES data was chosen for comparison because both studies are studies of biomonitoring of general population non-exposed occupationally to metals. Besides that the period of time of both studies is rather the same (2007–2008) stratified by the same age groups, allowing a good comparison between results. In our study the setting of Reference Values (RV) was done using the upper limit of the 95% confidence interval (95%CI) of the 95th percentile of all studied individuals. The reason for that is that the use of the upper limit of the distribution seems more appropriate to the evaluation of anybody suspected to be at high levels of exposure to a certain metal. Thus, a comparison was done with the 95th percentile result reported in the NHANES study. Multivariate analysis revealed that gender was one of the factors significantly associated with the blood lead levels. The BLL were higher among the men compared to the women. This finding may be accounted for by various factors, such as greater exposure to lead and higher hematocrit values among men and sex differences in lead metabolism (Batariová et al., 2006; Popovic et al., 2005; Skerfving et al., 1999; WHO, 1995). The BLL can increase parallel to the hematocrit because approximately 99% of the lead in blood is in the red blood cells (WHO, 1995). Smoking was a determinant factor for the blood lead levels. Our results indicate that the blood lead level was higher among smokers compared to nonsmokers. The lead content of cigarettes can vary from 2.5 μg/L to 12.2 μg/L, of which 2% to 6% may be inhaled by smokers

Table 6 Results of the regression analysis of the generalized linear model to assess the relationship between explanatory variables and blood lead concentrations. Factor

Coefficient

Standard error

N Minimum Maximum N b LOQ LOQ LOD Mean Mode N: number of samples; LOQ: limit of quantification; N b LOQ: number of values below LOQ; LOD: limit of detection.

Area of residence North Central East West South Gender Male Female Smoking Smoker Nonsmoker Ex-smoker Offal intake 5 or more times/week Up to 3 times/week

(95%) CI Wald Lower

Upper

p

0.27 2.31 −0.50 1.12 Ref.

0.40 1.10 0.33 0.85

−0.51 0.15 −1.14 −0.54

1.06 4.47 0.14 2.78

0.495 0.036 0.125 0.186

1.57 Ref.

0.30

0.99

2.15

b0.001

7.30 −0.90 Ref.

0.89 0.60

5.55 −2.07

9.05 0.27

b0.001 0.131

1.55 Ref.

0.59

0.39

2.72

0.009

C.S. Kira et al. / Science of the Total Environment 543 (2016) 628–635 Table 7 Results of the regression analysis of the generalized linear model to assess the relationship between explanatory variables and blood cadmium concentrations. Factor

Age range 11 years old or less 12 to 19 years old 20 years old or more Gender Male Female Smoking Smoker Nonsmoker Ex-smoker Distilled beverage intake At least once per week Up to 3 times/month

Coefficient

Standard error

(95%) CI Wald Lower

Upper

p

−0.073 0.036 Ref.

0.006 0.009

−0.085 0.019

−0.062 0.054

b0.001 b0.001

0.025 Ref.

0.004

0.017

0.033

b0.001

0.137 −0.156 Ref.

0.034 0.022

0.070 −0.200

0.205 −0.112

b0.001 b0.001

−0.048

0.019

−0.085

−0.010

0.013

Ref.

(Skerfving and Bergdahl, 2007; WHO, 1977; WHO, 1995). Depending on the particle size, solubility and chemical species, up to 50% of the inhaled lead may be absorbed (WHO, 1995). Several studies reported considerably higher BLL among smokers compared to nonsmokers, which may vary from 10% to more than 50% (Alimonti et al., 2005; Apostoli, 2002; Forte et al., 2011; Ikeda et al., 2011; McKelvey et al., 2007). In the present study, age exhibited a significant positive association with the BLL: the latter were higher among the older participants compared to the younger ones. Other studies also reported similar findings (Alimonti et al., 2005; Batariová et al., 2006; Ikeda et al., 2011; Koyashiki et al., 2010; Menezes Filho et al., 2012). Lead absorption is influenced by age. Among adults, approximately 10% of the ingested lead is absorbed, and absorption may be higher in the fasting condition or in the presence of dietary calcium, phosphate, selenium or zinc deficiency; among children, up to 50% of the ingested lead may be absorbed (WHO, 1995). The increase of the BLL parallel to age might also be explained by the fact that among adults, 94% of the lead present in the body is stored in the bones, while among children, this percentage is 73% (WHO, 1995). The lead accumulated in the bones can behave as an endogenous source of this metal, which might be mobilized into the bloodstream under some conditions, such as pregnancy, breastfeeding, menopause and aging, and in some physiological or pathological situations, such as osteoporosis and bone fractures, among others (Korrich et al., 2002; Lauwerys and Hoet, 2001; Popovic et al., 2005; WHO, 1995). Regression analysis model revealed that the participants' area of residence was associated with the BLL. The influence of this factor may be related to geographical differences, pointing out to different scenarios of exposure. Several studies have reported that the area of residence is a determinant factor for BLL, as it explains approximately 4% to 45% of its variance (Mattos et al., 2009; Menezes Filho et al., 2012; Pawlas et al., 2013; Schroijen et al., 2008). In the present study, offal intake was significantly associated with the BLL. Lifestyle-related factors, such as the diet, can influence BLL. Heavy metals tend to accumulate in the liver and kidneys; thus, these organs exhibit the highest levels of such elements compared to other edible animal parts, such as muscle. As a result, when those organs are consumed, they may behave as significant sources of exposure to metals (Obasohan, 2007). Few studies have investigated the relationship between offal intake and blood levels of metals. One study performed in Norway found a positive association between frequent offal intake and a slight elevation of the BLL (Birgisdottir et al., 2013). One limitation of our study is that the cadmium blood level was below the method's LOQ in 39% of the analyzed samples (Table 5). Therefore, as the proportion of values below the limit of quantification is high results for this metal should be viewed with caution.

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In Brazil, only one study sought to derive reference values of cadmium in blood. The population of that study consisted of 539 adult blood donors, who were recruited in the metropolitan area of São Paulo at the end of 2006. The reference value derived for non-smoking adults aged 18 to 65 years old was 0.6 μg/L (Kuno et al., 2013), while in our study, the reference value for non-smokers was 0.4 μg/L. The metropolitan area of São Paulo includes several industrial zones, which most likely contributes to the increase in the blood cadmium levels detected in the population of blood donors. The reference values of cadmium in blood derived for nonsmoking adults and children under 11 years old were lower compared to the ones established in the Czech Republic and Germany. The reference values derived for nonsmoking adults and children in the Czech Republic were 1.0 and 0.5 μg/L, respectively (Cerná et al., 2012). Based on data corresponding to the period from 1997 to 1999, the reference value of cadmium in blood established in Germany for nonsmoking adults was 1.0 μg/L (Wilhelm et al., 2004; Schulz et al., 2012). This value may be higher than the one derived in the present study because it was established approximately 10 years ago. No reference value was derived for children, but the German biomonitoring committee recommended the use of analytical techniques able to reliably confirm levels above 0.3 μg/L. Comparison to the 95th percentile of the blood cadmium levels in the population-based NHANES, conducted in the United States in 2007 and 2008 (1.60 μg/L for men, 1.43 μg/L for women and 0.90 μg/L for adolescents) (CDC, 2013), shows that the reference values derived in the present study are lower. In the NHANES, the 95th percentile of the blood cadmium levels for children 6 to 11 years old was 0.26 μg/L and was of the same magnitude of the one derived in the present study for children under 11 years old (0.2 μg/L) (Table 3). Differences in the exposure to cadmium among nonsmokers might be due to factors such as diet, water contamination, inhalation of polluted air and contact with contaminated dust, which may account for the increase in the blood cadmium levels (Nawrot et al., 2010). A study conducted in Korea with a representative sample using data collected by the population-based Korean National Health and Nutrition Examination Survey (KNHANES) in 2005 reported that the 95th percentiles of the blood cadmium levels were 3.01 μg/L for women and 2.97 μg/L for men above 20 years old (Kim and Lee, 2011). These levels are higher than the reference values derived in the present study for women and men (0.56 μg/L and 0.63 μg/L, respectively), which suggests that the Korean population is more exposed to cadmium compared to the population of São Paulo. In a study performed with Chinese children ranging from 1 to 12 years old, the reported median was 0.6 μg/L in both genders; the reference interval ranged from 0.5 to 3.0 μg/L for boys aged 11 and 12 years old and from 0.5 to 3.9 μg/L for girls in the same age range (Liu et al., 2012). The reference value derived for children under 11 years old in the present study (0.22 μg/L) was lower than the median level reported by Liu et al. (2012), indicating that Chinese children are exposed to some source of cadmium that should be investigated, as some areas of the country are contaminated by cadmium. The main nonoccupational sources of cadmium exposure are diet and smoking. In Asian countries, where rice is a staple food, rice should be monitored to control the dietary intake of cadmium (He et al., 2013). In Italy a study with adolescents aged 13 to 15 years in 2009 evaluated cadmium blood concentrations and the 95th percentile obtained for boys was 0.71 μg/L and 0.91 μg/L for girls (Pino et al., 2012). Comparing these results with the reference values derived in our study for adolescents aged 12 to 19 years (Table 3) one can observe that the values are similar for boys and lower for girls, pointing out that probably Italian adolescent girls are more exposed to cadmium than the Brazilians. In the present study, multivariate analysis revealed that age range, gender, smoking and intake of distilled beverages were significantly associated with blood cadmium concentrations. Relative to age, the reference values derived were higher for the older participants compared to

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the younger ones. This finding agrees with the results of other studies performed in the United Kingdom, Japan and Italy, which showed that the blood cadmium levels were positively associated with age (Ikeda et al., 2011; Madeddu et al., 2011; White and Sabbioni, 1998). According to the literature, cadmium accumulation in the body parallel to age is due to the lack of a biochemical mechanism of elimination combined with renal resorption of the metal (Madeddu et al., 2011; Roggi et al., 1995). The results of the present study further showed that blood cadmium concentrations were slightly higher among men compared to women. A study conducted in Italy with adults aged 20 to 79 years old also found that the blood cadmium levels were higher among men compared to women (Roggi et al., 1995). According to some reports in the literature, increased intestinal absorption of cadmium and, consequently, the blood levels of this metal are associated with iron and calcium deficiency. The results of a study performed in Sweden disagree with the ones of the present study, as the blood cadmium concentration was 1.4 times higher among women compared to men, the reason possibly being iron deficiency in the former (Madeddu et al., 2011; Olsson et al., 2002). Regarding the differences of blood cadmium levels according to sex, our results showed to be a little higher among men for the same age of women, in disagreement with the results reported by Lee and Kim (2014). According to these authors low ferritin levels and higher taxes of absorption of cadmium are the causes of higher cadmium levels in women blood compared to men. In our study we do not performed ferritin measurements to correlate with cadmium blood levels. Lack of iron status or menstruation status is a limitation of this study. Blood cadmium levels are typically higher among smokers (i.e., 1.5 to 10 times higher compared to nonsmokers) (Alimonti et al., 2005; Batariová et al., 2006; Becker et al., 2002; Cerná et al., 2012; Heitland and Köster, 2006; McKelvey et al., 2007; Mijal and Holzman, 2010; Nunes et al., 2010; Roggi et al., 1995; Son et al., 2009; White and Sabbioni, 1998; Wilhelm et al., 2004). In the present study, the blood cadmium concentration was 4 times higher among smokers compared to nonsmokers, which confirms the influence of cigarettes on the blood cadmium concentration. Inhalation is an important route of exposure to cadmium, especially in the case of smokers, due to the high rate of absorption of this metal via this route. On average, approximately 10% of the cadmium present in cigarettes is inhaled by smokers, and 5% to 35% of that amount is absorbed into the bloodstream (ATSDR, 2012; Chasin and Cardoso, 2003; Nordberg and Nordberg, 2002; Nordberg et al., 2007). Some studies show that smoking essentially doubles the cadmium body load (ATSDR, 2012). Blood cadmium levels seem to be influenced by alcohol consumption. In the present study, the blood cadmium concentration was almost 2-fold higher among the participants who consumed alcohol at least once per week, compared to the ones who drank up to three times per month. This finding agrees with the results reported by Forte et al. (2011), according to which the blood cadmium levels were 1.3-fold higher among Italians who consumed alcohol regularly compared to the ones who did not drink.

6. Conclusions In the present study, the blood lead and cadmium levels of adults, adolescents and children residing in the city of São Paulo were estimated, and the corresponding reference values were derived. The metal concentrations detected in the present study indicate that the exposure of the investigated population to lead and cadmium is low. The blood lead concentration was significantly associated with gender, smoking, offal intake, area of residence, and age. The blood cadmium concentration was significantly associated with gender, smoking, consumption of distilled beverages, and age.

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