Introduction

Epidemiological studies have provided strong evidence that exposure to environmental pollution represents a risk factor in the morbidity and mortality of several diseases, especially cancer (Arlandis and Valero 2012; Yang et al. 2016). Among the major abiotic agents causing stress to ecosystem, there are the toxic metals, which are largely used in industry and in agrotechnology, generating bioaccumulation through food chain (Maksymiec 2007; Anjum et al. 2012).

At this segment, tanneries and chrome plating factories contain a heterogeneous mixture of hazardous substances, emitting particles of elements such as chromium (Cr) in the environment (ATSDR 2012). The chrome plating process involves the addition of a coating of hexavalent chromium [Cr (VI)] on a metal piece. Thus, the worker involved in this process has a direct exposure to toxic metals. Besides the most important way to occupational exposure is inhalation (Fe’bel et al. 2011; Khan et al. 2012). However, the Cr (VI) is in the list of the 50 more hazardous substances of the Agency for Toxic Substances & Disease Registry (ATSDR 2012), and among the 20 priority substances from this institution (ATSDR 2011).

Chromium is a transitional metal with two main and stable valence forms: trivalent chromium [Cr (III)] and Cr (VI) (Barmo et al. 2006; Ciacci et al. 2012), which are also known as important elements at biological and industrial point of view (Papassiopi et al. 2014). This metal penetrates the human organism through lungs, gastrointestinal tract, and skin. Humans are commonly exposed to Cr through food intake, water drink, urban pollution air, hip and knee prosthesis, among others (ATSDR 2011; Gil et al. 2011; Ciacci et al. 2012). The absorption and retention of Cr depends on several factors: reduction rate of Cr (VI) to Cr (III) inside and outside the cells, the transport rate of Cr (VI) in cells, and the diffusion rate of Cr (III) from cells (NTP 1998). In general, independently of the route of exposure, the Cr (III) is less absorbed than Cr (VI), being the hexavalent form very toxic by dermic route and inhalation, causing several damages as lung cancer, nasal irritation, nasal ulcer, hypersensitivity reactions, and contact dermatitis (Fang et al. 2014; Silva and Pedrozo 2011; Zhang et al. 2011; Zhitkovich 2011).

Toxicological evidence indicates that the toxicity of Cr also appears to be related with oxidative stress, since Cr (VI) is reduced to Cr (III), generating Cr (V), and Cr (IV) inside the cells (Cervantes et al. 2001; Myers et al. 2011). The oxidative stress also involves the concentrations of glutathione (GSH), a great antioxidant that acts as an important defender of the organism against reactive oxygen species, and it is needed to prevent oxidation of proteins (Jomova and Valko 2011).

Furthermore, many studies have also indicated that exposure to this chemical in cell culture and animal models is associated with generation of oxidative stress and showed affinity by thiol groups of amino acids such as cysteine (Greenwood and Earnshaw 1997; Huang et al. 2004; Nunes-Tavares et al. 2005; Fillice et al. 2018). Due to this, it is believed that Cr exposure could interfere on activity of human thiol-containing enzymes such as pyruvate kinase (PK), creatine kinase (CK), adenylate kinase (AK), and δ-aminolevulinate dehydratase (ALA-D).

All these enzymes are fundamental to the human metabolism. Although studies evaluated Cr with involvement of oxidative stress, there is lack of investigation about the consequences of occupational exposure. In this context, the present study aimed to evaluate whether the biomarker of exposure to metals, especially chromium levels, presents associations with hematological and biochemical parameters and if these exposures can alter the activity of enzymes that contain thiol groups (PK, CK, AK, and ALA-D).

Materials and methods

Subjects

This study involved a total of 100 male participants. The exposed group consisted of 50 workers of two plating companies, from Rio Grande do Sul, Brazil, occupationally exposed to chromium. The control group consisted of 50 subjects that had administrative occupation and with no history of occupational exposure to chromium or other xenobiotics. Each subject answered a questionnaire interview in which information about individual characteristics, health status, history and lifestyle (smoking, alcohol drinking habits, diet, medication, exercise habits), and other general information regarding the work shift (years of service and time spent inside the car) was requested. The Committee of Ethics in Research of the Feevale University approved this study (project number 2.14.01.11.2242). An informed consent was obtained from all individual participants included in the study.

Sample collection

Biological samples

Urine and blood samples were collected from all participants at the end of the work shift following four consecutive days of exposure at the plating companies. Fifty milliliters of fresh urine was collected for the determination of metals and creatinine levels, stored at − 20 °C until the analysis. Blood venous samples from all the subjects were collected by venipuncture using Vacutainer® tubes containing EDTA (4 ml) and heparin (4 ml) anticoagulants. Aliquots from heparinized blood were transferred into Cornings® trace-metal-free centrifuge tubes and stored at 4 °C until analysis of metal levels. The remaining heparinized blood was stored at − 80 °C for PK, CK, AK, and ALA-D analyses.

Drinking water measurements

Approximately 500 mL of drinking water samples were collected from wells near to the two analyzed plating companies. Aliquots from water samples were used to ecotoxicological assay and other transferred to Cornings® trace-metal-free centrifuge tubes, previously acidified with 100 μL of ultrapure nitric acid. Tubes were properly identified and stored at 4 °C until determination of metal levels.

Allium cepa assay

The toxicological study in water samples was performed by analysis of the number of mutagenesis found in young roots of Allium cepa (common onion) according to Fiskesjö (1979). The protocol test consisted in the exposure of six onion bulbs for each sample for 24 h, at room temperature and in the absence of direct light. As negative control group, it was used mineral water.

Quantification of elemental concentrations in samples

In whole blood were measured non-essential metals chromium (Cr), vanadium (V), nickel (Ni), lead (Pb), and, the semimetal, arsenic (As). In serum, it was evaluated essential metals like iron (Fe), copper (Cu), zinc (Zn), and selenium (Se). For the measurement of element concentration in blood, urine, or water samples, 1 mL 65% ultrapure nitric acid was added to 500 μL of sample in a polypropylene digestion tube. Afterwards, the mixture was digested by heating at 95 °C for 4 h. Extracts were cooled at room temperature, and the volume was made up to 10 mL with ultrapure water. Toxic element concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS) using a NexIon 300X spectrometer (PerkinElmer-Sciex, USA) (Mateus et al. 2013). An Rh (400 μg/L) acidified aqueous solution (1% HNO3) was added in line as internal standard, and the calibration curve ranged from 5 to 80 μg/L. Calibration solutions were prepared using a 10 mg/L stock solution (PerkinElmer 29). The LOD and LOQ were calculated based on the standard deviation of the calibration blanks (n = 10): three times the standard deviation for the LOD (or ten times for the LOQ), divided by the slope of the calibration curve. Precision and accuracy of the analytical method were monitored with reference standards that were analyzed in intervals of 15 samples. For differences greater than 10%, a new calibration was applied. Accuracy of the method was evaluated by analysis of the certified reference material DORM-3 (National Research Council-Canada), performed by the same procedure employed for the samples. The concentration results agreed with the certified values, at a 95% confidence level, according to Student’s t test.

Hematological and biochemical parameters

Hematological parameters were measured on the ABX Micros 60 (Hematology Analyzer-Diamond Diagnostics, San Francisco, USA). Biochemical serum parameters evaluated were lactate dehydrogenase (LDH), to evaluate general tissues damages and creatinine, for kidney function. Alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured to evaluate liver function. These biochemical parameters were determined by dry chemistry on Vitros 950 (Johnson & Johnson, NJ, USA). The quantity of proteins contained in hemolyzed erythrocytes was performed by the Lowry et al. (1951), using bovine serum albumin as standard. In addition, creatinine in urine was also measured to provide chromium urinary concentration through calculation. The concentration was performed by spectrophotometry using commercial laboratory kits.

Determination of the activity of pyruvate kinase

The activity of PK in erythrocytes was determined using Leong’s method (Leong et al. 1981), with minor modifications. This procedure was performed in triplicate and prepared in microplate.

Determination of the activity of creatine kinase

The activity of CK in erythrocytes was measured according to Hughes (1962). This procedure was performed in triplicate and prepared in microplate.

Determination of the activity of adenylate kinase

The activity of AK in blood was measured according to Dzeja et al. (1999). This procedure was performed in triplicate and prepared in test tubes.

δ-Aminolevulinate dehydratase activity assay

ALA-D activity and index of reactivation (ALA-RE) were determined in heparinized blood by spectrophotometry according to Sassa (1982) with minor modifications. The reactions of this procedure were performed in duplicate and measured by spectrometry at 555 nm. ALA-D activity was expressed in U/L (nmol PBG h/mg Hb) and the indexes of reactivation expressed as percentage (%).

Erythrocyte levels of non-protein thiol groups

Levels of non-protein thiol groups were determined in erythrocytes by spectrophotometry at 412 nm, as described by Ellman (1959).

Statistical analysis

The analysis was performed using the Statistical Package for Social Sciences (SPSS) software. All study variables were tested for normality by the Shapiro–Wilk. Comparisons between groups were achieved by Student’s t test or Mann–Whitney U test. The results were expressed as mean ± standard deviation (SD). Correlation tests were performed according to Pearson’s correlation coefficient or Spearman’s rank according to each variable. Values of p ≤ 0.05 were considered significant.

Results

The data obtained from the questionnaire about the characteristics of the studied groups are summarized in Table 1. No significant difference was found regarding age between groups (p > 0.05). Sex distribution was not different between the two groups by Chi-Square analysis (p > 0.05). Age was distributed into four groups, and no difference was found between exposed and control groups by ANOVA (p > 0.05). Therefore, multiple models in the analysis of associations were not performed.

Table 1 Main characteristics of the study population
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The biomarkers used to evaluate the level of occupational exposure were Cr in blood, urine, and water. The blood concentration of Cr was significantly higher in the exposed group (2.02 ± 0.20 μg/L) when compared with control group (1.73 ± 0.16 μg/L) (p < 0.0001) (Fig. 1). Total Cr test was used as an indicator of exposure to Cr (VI) once there were no methodologies available to determine the concentrations of Cr (VI) in blood (Devoy et al. 2016). Similar results, but more expressive, were obtained about the quantification of Cr in urine. In the exposed group, the concentration was 10.65 ± 5.26 μg/g creatinine, whereas in the control group, was 4.05 ± 3.78 μg/g creatinine (p < 0.0001) (Fig. 2).

Fig. 1
figure 1

Blood chromium levels. Determination of blood chromium on exposed group (n = 48), 2.02 ± 0.20 μg/L, and control group (n = 50), 1.73 ± 0.16 μg/L. The data were analyzed by Student’s t test, t(97) = 7.09; **p < 0.0001 compared with controls. The data are expressed as mean ± standard deviation (SD).

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Fig. 2
figure 2

Urinary chromium levels. Determination of urinary chromium on the exposed group (n = 50), 10.65 ± 5.26 μg/g of creatinine, and control group (n = 45), 4.05 ± 3.78 μg/g of creatinine. The data were analyzed by Student’s t test, t(94) = 7.09; **p < 0,0001 compared with controls. The data are expressed as mean ± standard deviation

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In addition to Cr, other elements were evaluated in the blood samples. Concentrations of elements (essential and non-essential) in blood samples from the studied groups are presented in Table 2. Pb, As, Ni, and V levels in whole blood were increased in the exposed group compared to the control group (p < 0.001). Serum Fe, Zn, and Se levels were within normal ranges and there were no statistically significant differences between groups (p > 0.05). Cu levels were significantly high in exposed group when compared to controls (p < 0.001), but the levels were consistent with reference values.

Table 2 Concentration of elements (essential and non-essential) in blood of studied subjects
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Regarding Cr levels in drinking water, Cr concentrations in well water collected near to the two plating companies were 0.0010 mg/L and 0.0015 mg/L. Furthermore, the mean Cr concentrations registered were below the international standard established by the World Health Organization (0.05 mg/L) (WHO 2011), the US Environmental Protection Agency (EPA) (0.1 mg/L) (EPA 2009), and the Food and Drug Administration (FDA) (0.1 mg/L) (ATSDR 2012). It will be important, in continuing this work, to perform environmental surveillance such as the metal distribution in solution of plating and air in the workplace.

Additionally, ecotoxicological analyses were carried out with water samples using the plant system test Allium cepa. The results indicated the absence of damage cells, considering three parameters: mitotic index, micronucleus, and total of chromosomal abnormalities. About the mitotic index, the two spots of exposed group presented 3.2 ± 3.7% and 4.9 ± 2.5%, whereas the control group presented 5.5 ± 0.8%, (p > 0.05). The total of chromosomal abnormalities was 0.7 ± 1.3% and 0.7 ± 1.3% in the two samples of the exposed group, and 0.9 ± 0.9% in the control group (p > 0.05). None of the groups showed the presence of micronucleus (p > 0.05).

Hematological and biochemical parameters from both groups were within the reference values, according to Table 3. Despite that, hematological parameters (Hb, Ht, MCH, MCHC) and biochemical analysis (ALP, ALT, and LDH) were significantly increased in the exposed group than the controls (p < 0.05). Neither the hematological nor biochemical parameters were significantly associated with the biomarker of exposure.

Table 3 Hematological and biochemical parameters of the studied groups
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In relation to thiol-containing enzymes (PK, CK, and AK), all of them presented their activities statistically reduced on the exposed group when compared to the control group (p < 0.0001) (Table 4). Conversely, non-protein thiol levels were increased in exposed group (80.90 ± 23.43 μmol/mL of erythrocytes) when compared to the control group (46.70 ± 8.35 μmol/mL of erythrocytes) (p < 0.0001).

Table 4 Results of enzyme activities
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Exposed group had lower ALA-D activity than the controls (p < 0.001) (Table 5). Additionally, we evaluated the involvement of –SH groups in ALA-D inhibition investigating the effect of dithiothreitol (DTT), a –SH reducing agent, on the enzyme reactivation (ALA-RE with DTT). ALA-RE with DTT was significantly different between groups, with a recovery of 38.29 ± 22.81% exposed group vs. 18.42 ± 8.83% in the control group (p < 0.0001). Moreover, the ALA-D reactivation was also investigated with the addition of ZnCl2 (ALA-RE with ZnCl2), demonstrating that whether zinc ions protect and/or restore the ALA-D inhibition caused by metals. ALA-RE with ZnCl2 was significantly different between groups, with a recovery of 26.69 ± 30.09% exposed group vs. 10.53 ± 5.31% in the control group (p < 0.0001).

Table 5 Results of ALA-D activity and reactivation index
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The effects of dose dependence of Cr levels versus thiol-containing enzymes were analyzed by linear regression and are displayed in Table 6. The same ascertainment was performed to evaluate dose dependence of some other elements (essential and non-essential) versus thiol-containing enzymes, as seen in Table 7 for PK, CK, and AK, and in Tables 8 and 9 for ALA-D.

Table 6 Regression models of the association between chromium and enzymes: pyruvate kinase, creatine kinase, adenylate kinase, and ALA-D activity/reactivation index
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Table 7 Regression models of the association between non-essential/essential metals or semimetal and enzymes: pyruvate kinase, creatine kinase, and adenylate kinase
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Table 8 Regression models of the association between metals or semimetal and enzyme ALA-D, considering DTT
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Table 9 Regression models of the association between metals or semimetal and enzyme ALA-D, considering ZnCl2
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Discussion

Biomonitoring has been used as a tool in occupational settings, in the assessment of exposure and surveillance of workers exposed to hazardous chemicals, such as metals. These metals are involved in the etiology and prognosis of many illnesses. To the best of our knowledge, this is the first report that investigates the influence of the occupational exposure to chrome plating on activity of enzymes that contain thiol groups (PK, CK, AK, and ALA-D), hematological and biochemical parameters. Considering that people may spend long time in the workplace, occupational exposure to xenobiotics is becoming an important public health problem (Faisandierl et al. 2011).

It is known that chromium, in its hexavalent form, affects thousands of workers through occupational exposure. Likewise, the damages generated by this element are extensive, causing pathologies such as cancer, dermatitis, and metabolism modifications, as in the oxidative stress process. Moreover, our findings are indicative that this occupational exposure is interfering to human health, corroborating with previous studies (Zhang et al. 2011). The International Agency for Research on Cancer classifies Cr (VI) as group 1 (considered carcinogenic to humans) (IARC 2018).

In relation to environmental evaluation, it was observed that, although the industry process reaches workers beings, the hydric environment did not seem to be significantly changed in this analysis. In addition, the Allium cepa test, considered a fast and cheap technique with great sensitivity on detection of environmental chemical agents (Leme and Marin-Morales 2008), did not present differences between water samples collected at chrome plating companies and control negative.

The concentration values of blood and urinary Cr are in the range of safe values, being the reference value < 3 μg/L for blood Cr (Zeisler and Young 1987) and, according to the Brazilian program of medical control of occupational health, a maximum Cr concentration of 5 μg/g of creatinine in urine (MTE 1994). Since only Cr (VI) can penetrate the blood cells, differently from Cr (III), it is estimated that the total Cr test is the indicator of Cr (VI) exposure (Devoy et al. 2016). However, our data showed significant differences in the urinary chromium concentration between exposed workers and control group. This biomarker was elevated in the exposed group when compared with controls, making this parameter a more sensitive evaluation in terms of recent exposure, whereas the blood Cr can be used as an indicator of long-term exposure (Gil et al. 2011). These results reinforce the importance of recommended actions by the American Conference of Governmental Industrial Hygienists (ACGIH 2004), such as a maximum of 8 h per work shift, avoid contact of skin with the metal, use the personal protective equipment, to protect eyes, skin, and airways, as well as the air monitoring on the work environment. These steps can prevent a contamination by chromium in the workers. The lack of personal protective equipment was evidenced among all the workers of this study, which can be one of the factors that led to a dangerous chromium exposure.

Additionally, to Cr, other elements were evaluated in the blood samples of workers. Exposed group presented significant increases of Pb, As, Ni, and V levels compared to controls. Furthermore, Ni and V levels were above the limits recommended by WHO (WHO 1996). These results pointed to a work environment with a large quantity of hazardous substances, which some of them are even recognized as toxic to thiol-containing enzymes activity as well as participants of metal-induced oxidative stress (Baierle et al. 2014; Feksa et al. 2012; Jomova and Valko 2011). Toxicological studies in vitro suggested that these metals elicit oxidative stress by inducing the generation of reactive oxygen species (ROS), decreasing the activities of cellular antioxidant enzymes, leading to damage of macromolecules (Nunes-Tavares et al. 2005; Fillici et al. 2018).

Additionally, biomonitoring of elements considered essential is also very important for occupational health, mainly because these are required for normal physiological function and are involved in numerous biochemical mechanisms. In this context, our results showed that concentrations of essential metals Zn, Fe, and Se are almost the same between exposed and non-exposed individuals, proving that their lifestyle, above all the alimentation, is quite similar. The exception is the Cu, which can be explained by the fact that plating companies utilize in their processes compounds of several metals, which could include Cu. So, these workers would be occupationally exposed to this metal, increasing the physiological levels.

Historically, the hemogram and biochemical parameters have been used as a simple, readily available screening tool for assessing of effect biomarkers in occupational exposure. These results were within the reference value, which concludes that they are not effective to use as biomarkers of occupational exposure.

The PK is the most important enzyme in glycolysis, including in erythrocyte, which do not have oxidative phosphorylation to produce adenosine triphosphate (ATP), essential compound to generate energy to the erythrocyte. The PK catalyzes phosphotransferase of a PEP’s phosphate to adenosine diphosphate (ADP), generating ATP and pyruvate (Dalpino et al. 1998). The creatine kinase has the main function in energy metabolism, where it works like a buffer system of levels of cellular ATP. The CK catalyzes the reversible transfer of the phosphoryl group of phosphocreatine to ADP, regenerating ATP (Warchala et al. 2006). Already, the adenylate kinase is responsible for performing the reversible conversion between phosphoryl of ATP, ADP, and AMP (Bae and Phillips 2006). The AK doubles the energy potential of ATP, having the ability to regenerate ATP from two ADP and by the regulation of processes involving adenine nucleotides (Noma 2005; Willemoes and Kilstrup 2005).

Although the Cr concentrations are in the expected range, the exposed workers presented a significantly decrease of thiol-containing enzyme activity. The decrease of PK, CK, and AK activity can generate innumerous damages in erythrocyte metabolism, primarily to energy regulation, since these enzymes are largely involved with the production, transference, and balance of nucleotides as ADP, AMP, and ATP, as well as control of generation of important metabolites as, for example, pyruvate (Akagi et al. 2006; Fothergill and Michelis 1993; Warchala et al. 2006).

In the current study, we also quantified non-protein thiol, which are considered the major endogenous antioxidants. Our results revealed that the exposed group presented increased levels compared to controls. Elevation of non-protein thiols suggests a compensatory mechanism to protect against the accumulation of reactive species, since the exposure of hazardous elements can induce an imbalance between effectiveness of antioxidant activity and generation of reactive oxygen species (Brucker et al. 2013).

The ALA-D is the most sensible enzyme of the heme pathway in erythrocytes, catalyzing the biosynthesis of porphobilinogen with two molecules of aminolevulinic acid (Akagi et al. 2006; Schubert et al. 2009). These enzymes in zinc-dependent and thiol groups are essential to its activity (Bernard and Lauwerys 1987). The analysis of ALA-D demonstrated that the Cr also decrease the activity on this enzyme. On the other hand, the index reactivation of ALA-D on the DTT and ZnCl2 evaluation was higher in the exposed group, probably because the thiol groups of this zinc-dependent enzyme are inhibited due to action of oxidant agents. The DTT is a substance with the capacity of reduced –SH groups, being capable of, in vitro procedures, to prevent or to revert a decrease of ALA-D activity caused by oxidants agents (Valentini et al. 2007). In this study, it was evidenced a higher reactivation index of ALA-D in the exposed group, compared to the control group, with the DTT, proving the action of oxidant agents in the metabolism of exposed workers. Similar data were found regarding to ZnCl2. The ALA-D is a thiol enzyme that depends on Zn (II) ions to have a correct operation (Sauer et al. 2014). The values obtained in this study pointed to a higher recovering of ALA-D activity with the presence of ZnCl2, proving the importance of this component in the active site of this enzyme.

In the same way, the increase of hematimetric indices in exposed group can indicate an attempt of the organism to compensate the heme pathway, impaired by the decrease of activity and efficacy of ALA-D. Similarly, several studies suggest that exposure to Cr is an important factor that leads to serious damage, including to metabolism, organs, and even cognitive impairments (Cao et al. 2014; Nascimento et al. 2015). These facts are observed by the higher concentration of LDH in the exposed group, suggesting a natural hemolysis process and/or tissue damage, and also as the increase of hepatic enzymes indicates that the exposure to heavy metal, such as Cr (VI), can induce injuries to human organs, being the liver affected in the exposed workers of this study.

After the linear regression study, it was verified that there is a relation dose dependent between Cr levels and activity of all the thiol-containing enzymes analyzed in this study, indicating that the higher the concentration of blood Cr, the lower the enzyme activity. Similarly, the metals V, As, Ni, and Pb also showed a presence of a relation dose dependent with all these enzymes. The regression analysis of Cu indicated that it could interfere only on the activity of AK. Therefore, we can also assign the injury caused to enzymes studied due to increased presence of metals Pb, As, Ni, V, and Cu in exposed individuals.

This reduction of activity of all enzymes could be the result of the capacity of Cr of binding itself on thiol groups of enzymes, reducing their activities (Nunes-Tavares et al. 2005). As is known, some thiol-containing enzymes have their activities, in erythrocytes, changed by thiol/disulfide exchange between sulfhydryl groups and disulfides of proteins (Gilbert 1984). Moreover, other metals assessed in this study may also change the functioning of enzymes like AK, CK, PK, and ALA-D.

In conclusion, our results indicate an important association between occupational exposure in chrome plating, with decreased activity of enzymes that contain thiol groups (PK, CK, AK, and ALA-D). This study has also provided important evidence that, in addition to Cr exposure present in environmental chrome plating other metals could be present and which may be related to changes in effect biomarkers evaluated in this study. It suggests that besides the presence of other metals, Cr demonstrated to influence the activity of the enzymes analyzed in this research.