Gender differences in cadmium and cotinine levels in prepubertal children

Highlights

• In 216 children from 6 to 11 years old the median level of Cd was 0.18 µg/L.

• The median level of cotinine was 1.50 µg/L.

• Correlation between levels of Cd and creatinine in all children was detected.

• Cd levels were different between 6–7 year old, 10–11 year old girls vs. 11 year old boys.

Abstract

Susceptibility to environmental stressors has been described for fetal and early childhood development. However, the possible susceptibility of the prepubertal period, characterized by the orchestration of the organism towards sexual maturation and adulthood has been poorly investigated and exposure data are scarce. In the current study levels of cadmium (Cd), cotinine and creatinine in urine were analyzed in a subsample 216 children from 12 European countries within the DEMOCOPHES project. The children were divided into six age–sex groups: boys (6–8 years, 9–10 years and 11 years old), and girls (6–7 years, 8–9 years, 10–11 years). The number of subjects per group was between 23 and 53. The cut off values were set at 0.1 µg/L for Cd, and 0.8 µg/L for cotinine defined according to the highest limit of quantification. The levels of Cd and cotinine were adjusted for creatinine level. In the total subsample group, the median level of Cd was 0.180 µg/L (range 0.10–0.69 µg/L), and for cotinine the median wet weight value was 1.50 µg/L (range 0.80–39.91 µg/L). There was no significant difference in creatinine and cotinine levels between genders and age groups. There was a significant correlation between levels of cadmium and creatinine in all children of both genders. This shows that even at such low levels the possible effect of cadmium on kidney function was present and measurable. An increase in Cd levels was evident with age. Cadmium levels were significantly different between 6–7 year old girls, 11 year old boys and 10–11 year old girls. As there was a balanced distribution in the number of subjects from countries included in the study, bias due to data clustering was not probable. The impact of low Cd levels on kidney function and gender differences in Cd levels needs further investigation.

Introduction

Prepuberty and puberty are critical periods of maturation during which hormones orchestrate the development of secondary sexual characteristics, skeleton and behavior. Estrogen and testosterone levels during these periods may be disturbed by environmental stressors from air, water, and food. A large number of xenobiotics/xenoestrogens couple estrogen receptors at different efficiency, thus redirecting biological pathways of maturation. The fact that some agents such as cotinine and fungicides act as aromatase inhibitors by lowering levels of estrogen and increasing levels of testosterone (Barbieri et al., 1986, Trösken et al., 2006) has not been investigated to the same extent. The currently reported increased incidence of early maturation or delay in onset of puberty in industrialized countries may have been related to environmental settings contaminated with hormonally active agents (Jacobson-Dicman and Lee, 2009). Additionally, exposure to endocrine disruptors during prepuberty may have significant implications on health risks during adulthood such as an increase in sterility of both genders, as well as increased incidence of testicular cancer and higher incidence of breast cancer in younger age groups (Fucic et al., 2012, Giannandrea et al., 2013, Toppari and Juul, 2010, Znaor et al., 2013).

Estrogen is defined as an endocrine and paracrine agent and neuromodulator that acts via three receptors so far known. By interacting with receptors xenoestrogens may have broad biological effects on the human organism. The impact of endocrine active agents on carcinogenesis is of great significance as according to current knowledge all cancers are accompanied by disturbances in estrogen receptor distribution and/or estrogen and aromatase levels (Fucic et al., 2012; Fucic et al., 2010; Yakimchuk et al., 2012; Okitsu et al., 2010; Kim et al., 2013). However, xenoestrogenic effects are not limited to carcinogenesis since other systems like the cardiovascular system and the urinary system are also modulated by estrogen (Hagenfeldt and Eriksson, 1988, Robinson et al., 2013). Also, disturbances of androgen receptors and testosterone levels are related with for example breast, lung and prostate cancer (Thike et al., 2014, Chang et al., 2013).

Cadmium is one of the most common endocrine disruptors in a child’s living environment which may be present in food and air (due to environmental tobacco smoke, ETS) (Satarug and Moore, 2004, Watanabe et al., 2013). Rice is the most frequent dietary source of Cd (Kipppler et al., 2012). Furthermore, the dietary intake of toxic metals including Cd may be higher in children than in adults (Liu et al., 2010).

Cadmium is a well-known toxic metal that binds to estrogen receptors (metalloestrogen) (Buzard and Kasprzak, 2000, Byrne et al., 2013, Crespo-López et al., 2009, Darbre, 2006; Nasiadek et al., 2011). This toxic metal may cause different neurotoxic effects, but also inflammation which is associated with Cd-related cardiovascular diseases and oxidative stress (Colacino et al., 2014).

The benchmark dose for Cd in adults for renal effects has been calculated to be 0.6–1.1 µg/g creatinine (Suwazono et al., 2006). A recent study in children showed that exposures even below the reference level for Cd based on renal effects, set by the European Food Safety Authority to 1 µg/g creatinine and to 5.24 µg/g creatinine by to the World Health Organization, still cause learning disabilities (Ciesielski et al. 2012). In an adult population of Swedish women tubular and glomerular effects in the kidney were already significantly increased at mean levels of 0.8 µg/g creatinine (Akesson, et al., 2005). A long term study conducted in Japan suggested irreversible and slowly progressive renal tubular dysfunction after exsposure to environmental Cd even after reduction of exposure (Iwata et al., 1993). Additionally, Cd exposure has been associated with a decrease of estradiol levels (Zhang et al., 2008). The specific susceptibility of the prepubertal period for toxic metals has been shown in an animal model, where exposure to lead until puberty caused higher renal toxicity than exposure during adulthood (Berrahal et al., 2011).

Metallothioneines (MT) are metal binding proteins present in humans in four isoforms and regulated by hormones. The complex of Cd and MT is nephrotoxic (Klaassen et al., 2009) and its bioaccumulation during prepuberty and puberty, as well as its specific gender related susceptibility, has not yet been investigated.

Apart from Cd, nicotine is another common pollutant present in the life environment of children, and potentially has an impact on the developing hormone system. Environmental tobacco smoke has mainly been known to be associated with an increased incidence of asthma, wheezing illnesses, bronchiolitis and cognitive abilities even at very low levels (Hwang et al., 2012, Tung et al., 2013, Yolton et al., 2005). Despite global efforts for reducing the number of smokers, children are not protected as they are predominantly exposed at home. Cigarette smoke is a source of both chemical agents and radiation (polonium). Cotinine, a metabolite of nicotine, is a commonly used biomarker of exposure to tobacco smoke. However, cotinine is also an active molecule that can have impact on human health via several mechanisms. Additionally to aromatase inhibition (an anti-estrogen activity) (Barbieri et al., 1986, Kadohama et al., 1993; Myong et al., 2013; Soldin et al., 2011; Zeller and Berger, 1989), cotinine is mitogenic for smooth muscle cells at concentrations seen among passive smokers. This represents a potential risk for blood vessel intimal hyperplasia (Calphor et al., 1997). Cotinine has also been shown to be an immunosuppressor in animal models, but whether this effect is a consequence of cotinine or testosterone, which is also known to be immunosuppressive (Furman et al., 2013), is unknown. An increase of testostosterone by aromatase inhibition has not been proven (Kalra et al., 2004). Exposure to ETS decreases levels of estradiol in women (Soldin et al., 2011).

The prepubertal period may be a vestibule for puberty and thus crucial in the final physiological preparation for puberty, which therefore makes it specifically vulnerable to the environmental contaminants (Mantovani and Fucic, 2014). The possible impact of hormonally active agents from the living environment on the prepubertal period has still not been investigated.

The COPHES and DEMOCOPHES twin projects, which represent the first European harmonized biomonitoring study on children and their mothers in 17 countries, included measurements of urinary cadmium (Cd) and cotinine. Children were the primary target group of the study because the EU Environment and Health Action Strategy (SCALE) was focused on children for their particular and universally recognized vulnerability (Becker et al., 2014; Den Hond et al., 2015; Casteleyn et al., 2015).

The aim of the current study was to investigate possible age and gender differences in levels of Cd and cotinine between the prepubertal period defined as age 6–7 and 6–8 years for girls and boys, respectively (Berberoglu, 2009, Golub et al., 2008) and the early pubertal period. In the study a subample of COPHES/DEMOCOHES cohort was analyzed. As this is the first study to compare the possible biological effects of Cd and cotinine during the prepubertal and early pubertal period, it was not possible to refer to any already agreed criterion in regard to the selection of age groups and the investigated biomarkers, thus the age groups were selected arbitrarily taking into account that girls enter puberty before boys.

Although most studies investigate ETS in children using questionnaires it has been shown that the method is unreliable as smokers may not report their smoking habits acurately. Therefore, cotinine levels in urine are a much more objective measurement (Butz et al., 2011). In the current study Cd was measured in urine as a reliable biomarker of exposure (Trzcinka-Ochocka et al., 2004). As increased creatinine clearance has recently been shown to be a good indicator of tubular damage in populations environmentally exposed to Cd (Kobayashi et al., 2008) a correlation analysis of Cd and creatinine was performed. Additionally, impact of Cd on levels of creatinine was investigated and compared between selected gender and age groups.