# Biological Monitoring of Hexavalent Chromium and Serum Levels of the Senescence Biomarker Apolipoprotein  J/Clusterin in Welders

Authors: Evangelos C. Alexopoulos; Xenophon Cominos; Ioannis P. Trougakos; Magda Lourda; Efstathios S. Gonos; Vassilios Makropoulos
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

Welding fumes contain metals and other toxic substances known or strongly suspected to be related with oxidative stress and premature cellular senescence. Apolipoprotein J/Clusterin (ApoJ/CLU) is a glycoprotein that is differentially regulated in various physiological and disease states including ageing and age-related diseases. In vitro data showed that exposure of human diploid fibroblasts to hexavalent chromium (Cr(VI)) resulted in premature senescence and significant upregulation of the ApoJ/CLU protein. In this study we analyzed blood and urine samples from shipyard industry welders being exposed to different levels of Cr(VI) over a period of five months in order to assay in vivo the relation of ApoJ/CLU serum levels with Cr(VI). Our findings confirmed the previously reported in vitro data since reduction of Cr levels, after a worksite intervention, associated with lower levels of ApoJ/CLU serum levels. We concluded that the human ApoJ/CLU gene is responsive to the acute in vivo oxidative stress induced by heavy metals such as hexavalent chromium.


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## Body

## 1. Introduction

Welders are
exposed to many air contaminants such as iron oxide, manganese, nickel, cadmium
oxide, zinc oxide, chromium, fluoride, ozone, nitrogen oxides, carbon monoxide,
and others [1]. Previous studies suggested that the increased generation of
highly reactive oxygen species, which result in oxidative tissue damage, is
responsible for the toxicity of Cr(VI), Fe, Ni, and other metals [2–6]. Welding
processes, like manual metal finishing, are commonly used in stainless steel
welding and produce mainly chromium, nickel, manganese, fluorides, nitrogen
oxide, and ozone [1].The
genotoxic, mutagenic, and cytotoxic effects of hexavalent chromium (Cr(VI))
exposure are well documented [2–6]. Chromium is
absorbed via the gastrointestinal and respiratory tracts and the skin. Even though
chromium kinetics is not fully clear, key features mainly include differential
absorption of Cr(VI) and Cr(III), rapid reduction of Cr(VI) to Cr(III) in all
body fluids and tissues, modest incorporation of chromium into bone, and concentration-
dependent urinary clearance [7]. Compared to the Cr(III) ions that cross the
membranes slowly by simple diffusion, Cr(VI) readily crosses cell membranes in
the form of tetrahedral chromate anions through the general anion transport
system and for that reason intracellular Cr is considered as indicative of
Cr(VI) exposure [8–10]. Inside
the cell, Cr(VI) is reduced to Cr(III), generating intermediate Cr(V) and
Cr(IV) ions, oxygen, and organic radicals. The existing evidence points to
Cr(V) as the main reactive species in Cr(VI)-induced genotoxicity [11, 12]
through direct redox reactions with DNA, formation of DNA adducts,  and Zn(II) thiolate complexes [13] which, in addition to Cr(VI) complexes formed mainly with
cellular thiols, are likely triggers of a chain of events leading to
carcinogenesis [14]. Products including DNA strand breaks, Cr-DNA adducts, and
DNA-protein cross-links have been shown to occur in vivo and in vitro [15, 16].The
distribution of chromium to different compartments, the possibility of
different transport mechanisms and pathways combined with the potential for
reduction of Cr(VI),
complicates further the kinetic  models as far as it concerns
excretion [17, 18]. The decrease of Cr(VI) levels from body fluids seem to
follow a biphasic blood clearance and a bi/multiphasic urinary excretion
pattern, which suggest the existence of several slow-releasing storage
compartments. Several studies have shown half-times ranging between a few days
(2–6) up to more
than 3 months or even two years for the fast/medium and slow phase elimination,
respectively [19–21].Recently
reported data suggested that exposure of human diploid fibroblasts to
hexavalent chromium Cr(VI)
at concentrations equal or 10 fold lower than the maximum permissive values
(MPV) resulted in cell death or premature cellular senescence, respectively
[22]. The cellular senescence phenotype was accompanied by elevated protein
levels of apolipoprotein J/clusterin (ApoJ/CLU) [22].Human ApoJ/CLU
is a heterodimeric secreted glycoprotein that was initially purified from serum
and identified as an apolipoprotein [23]. Not only ApoJ/CLU functions as an
apolipoprotein, but it is also implicated in additional intra-or extracellular
processes. For instance, it has been proposed that the secreted ApoJ/CLU
protein functions as an extracellular chaperone [24]. CLU is differentially
regulated in many severe physiological disturbance states including ageing,
several neurological diseases, and in vivo cancer progression [23].Interestingly
and surprisingly, in a cross-sectional field survey, it has been also found
that welders and sandblasters (known to be exposed to high levels of heavy
metals and other chemicals) exhibited lower ApoJ/CLU serum levels as compared
to other low chemically exposed occupational groups like white collars and
electricians [22]. Given these observations in this study we analyzed blood and
urine samples from shipyard industry welders being exposed to different levels
of Cr(VI) over a period of five months in order to assay in vivo the
relation of ApoJ/CLU serum levels with Cr(VI).


## 2. Materials and Methods

### 2.1. Sample Collection

Blood and urine samples were collected from male welders (n=75) and
sandblasters (n=5) of a shipyard industry according to standard procedures.
Subjects aged between 22 to 58 years old (mean 40.14) had worked for 2 to 35
years in the shipyard (mean 18.5) and agreed to participate in this study after
signing an informed consent. The male welders examined in this study were
selected among welders who welded on both MS and SS. Welding has taken place
inside workshops or SS tanks. Welders used electrodes containing various metals
in different concentrations like Mn (0.8–6.5%), Ni (0.02–8.8%), Cr
(0.03–22.5%), Mo, Si, Fe, Zn, Cu, and other substances. All welders had access
to local suction at their workplace and used it more than 75% of the welding
time. None wore airstream helmet, but all occasionally wore filter mask for
personal respiratory protection and consistently used protective clothing and
gloves. Each participant completed a comprehensive questionnaire on individual
welding history and on welding methods and intensity applied during the
previous two weeks, month, and the previous year. They were also asked about
their welding employment histories
and the duration of SS welding in their careers. As determined after detailed medical
examination, none of the subjects suffered any serious chronic disease.All welders
with Cr blood levels above2μg/L (n=9) were selected to
enter an intervention phase. This worksite intervention aimed to lower Cr(VI)
exposure through a minimization of stainless steel welding. Five months later,
sample collection was repeated in this selected group of workers. The five
months period was selected based on half time of chromium life and life cycle of erythrocytes.In parallel
to blood and urine collection, a number of additional parameters were recorded
such as age, anthropometrical characteristics, smoking status, duration of
employment, medical history, and detailed occupational history.Measurement of Cr and ApoJ/CLU levels in blood   and
urine samples
Cr levels in blood and urine samples were measured by using a Perkin-Elmer
600 atomic absorption spectrometer. Samples were appropriately diluted (1/2 for
urine and 1/5 for blood) with Triton X-100 in a nitric acid solution. They were
then analyzed by the standard addition calibration procedure using graphite
furnace [25] at a detection limit of 0.1μg/L. to correct the differences
in fluid intake, the urinary values were also related to the respective
creatinine values. Quantitative measurement of ApoJ/CLU serum levels by ELISA
was performed as described previously [22]. Additional biological parameters
assayed included γ-glutamyltranspeptidase (γ-GT; an indicator of liver function), alanine
aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol,
triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL),
urea nitrogen, uric acid, fasting glucose, erythrocyte sedimentation rate at
one hour, white blood cell count and type, platelet count, and hemoglobin.
Prostate specific antigen (PSA) level was determined in subjects aged above 45
years old. The biochemical parameters were measured by using a Hitachi
917 analyzer and the hematological
parameters at an autoanalyzer Pedra 120.


### 2.2. Statistical Analysis

Results were
expressed as mean (SD) or geometric mean (minimum-maximum). Differences between
groups were examined by Student’st-test or Mann-Whitney U-test depending on
the normality of the distribution. Pearson correlation analysis was used to
determine possible correlations between variables. A log transformation was
used for laboratory variables not fitting to a normal distribution. Wilcoxon
signed rank test was employed to assess the difference on paired observations
in intervention group. For comparisons, the two-tailed test was used with a
type I error of α=0.05. Data analyses were
conducted by means of the SPSS for Windows 14.1.0 statistical package.


## 2.1. Sample Collection

Blood and urine samples were collected from male welders (n=75) and
sandblasters (n=5) of a shipyard industry according to standard procedures.
Subjects aged between 22 to 58 years old (mean 40.14) had worked for 2 to 35
years in the shipyard (mean 18.5) and agreed to participate in this study after
signing an informed consent. The male welders examined in this study were
selected among welders who welded on both MS and SS. Welding has taken place
inside workshops or SS tanks. Welders used electrodes containing various metals
in different concentrations like Mn (0.8–6.5%), Ni (0.02–8.8%), Cr
(0.03–22.5%), Mo, Si, Fe, Zn, Cu, and other substances. All welders had access
to local suction at their workplace and used it more than 75% of the welding
time. None wore airstream helmet, but all occasionally wore filter mask for
personal respiratory protection and consistently used protective clothing and
gloves. Each participant completed a comprehensive questionnaire on individual
welding history and on welding methods and intensity applied during the
previous two weeks, month, and the previous year. They were also asked about
their welding employment histories
and the duration of SS welding in their careers. As determined after detailed medical
examination, none of the subjects suffered any serious chronic disease.All welders
with Cr blood levels above2μg/L (n=9) were selected to
enter an intervention phase. This worksite intervention aimed to lower Cr(VI)
exposure through a minimization of stainless steel welding. Five months later,
sample collection was repeated in this selected group of workers. The five
months period was selected based on half time of chromium life and life cycle of erythrocytes.In parallel
to blood and urine collection, a number of additional parameters were recorded
such as age, anthropometrical characteristics, smoking status, duration of
employment, medical history, and detailed occupational history.Measurement of Cr and ApoJ/CLU levels in blood   and
urine samples
Cr levels in blood and urine samples were measured by using a Perkin-Elmer
600 atomic absorption spectrometer. Samples were appropriately diluted (1/2 for
urine and 1/5 for blood) with Triton X-100 in a nitric acid solution. They were
then analyzed by the standard addition calibration procedure using graphite
furnace [25] at a detection limit of 0.1μg/L. to correct the differences
in fluid intake, the urinary values were also related to the respective
creatinine values. Quantitative measurement of ApoJ/CLU serum levels by ELISA
was performed as described previously [22]. Additional biological parameters
assayed included γ-glutamyltranspeptidase (γ-GT; an indicator of liver function), alanine
aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol,
triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL),
urea nitrogen, uric acid, fasting glucose, erythrocyte sedimentation rate at
one hour, white blood cell count and type, platelet count, and hemoglobin.
Prostate specific antigen (PSA) level was determined in subjects aged above 45
years old. The biochemical parameters were measured by using a Hitachi
917 analyzer and the hematological
parameters at an autoanalyzer Pedra 120.


## 2.2. Statistical Analysis

Results were
expressed as mean (SD) or geometric mean (minimum-maximum). Differences between
groups were examined by Student’st-test or Mann-Whitney U-test depending on
the normality of the distribution. Pearson correlation analysis was used to
determine possible correlations between variables. A log transformation was
used for laboratory variables not fitting to a normal distribution. Wilcoxon
signed rank test was employed to assess the difference on paired observations
in intervention group. For comparisons, the two-tailed test was used with a
type I error of α=0.05. Data analyses were
conducted by means of the SPSS for Windows 14.1.0 statistical package.


## 3. Results

One out of three welders
had a normal body mass index (BMI) (20–25 kg/m2), while 18.2% were
obese (BMI>30 kg/m2). Only 19.1% of the subjects were nonsmokers
and statistical analysis revealed a significant positive correlation between
cigarette smoking and elevated triglyceride and hematocrit levels. Body mass
index (BMI) besides age was also positively correlated to triglyceride level,
uric acid, and γ-GT (Table 1). Welders had
been welding for 11–25 days the
previous month and for 180–240 days the
previous year.Table 1
Individual
characteristics and laboratory results in shipyard workers.
SS weldersSandblastersWeldersn = 47n = 5n = 28Mean  sdMean  sdMean  sdAge (years)40.4 (9.0)46.8 (6.6)38.6 (10.8)Duration of employment (years)17.4 (11.5)26.3 (7.8)18.8 (12.6)BMI (kg/m2)29.1 (3.7)28.0 (1.7)26.5 (2.6)Glucose (mg/dL)89.2 (12.4)98.4 (11.0)85.0 (12.1)Urea (mg/dL)36.0 (8.8)40.6 (10.3)31.1 (6.7)Creatinine1.0 (0.1)0.9 (0.1)0.9 (0.1)Uric acid (mg/dL)5.5 (1.3)5.0 (0.2)5.4 (0.9)Liver function indicatorsALT (GPT) (U/L)36.3 (18.4)29.6 (12.1)29.3 (13.2)AST (GOT) (U/L)22.7 (7.7)18.4 (3.1)21.0 (6.1)GGT (U/L)31.4 (15.5)24.6 (6.4)25.7 (17.4)Lipid metabolismCholesterol (mg/dL)216.7 (43.8)200 (35.3)207.2 (35.8)LDL (mg/dL)138.4 (44.4)131.2 (29.6)134.7 (31.8)HDL (mg/dL)47.7 (8.5)44.2 (4.6)46.2 (7.9)Triglycerides (mg/dL)143.0 (122.7)122.6 (53.7)133.3 (54.1)HematologyHematocrit (%)45.0 (2.6)45.6 (3.3)45.4 (2.7)ESR (mm)6.8 (5.6)7.80 (7.1)3.6 (2.0)Leukocytes (WBC) (103)7.51 (2.08)7.57 (0.55)8.10 (1.99)Neutocytes (103)4.23 (1.64)4.29 (0.26)4.62 (1.40)Lymphocytes (103)2.63 (0.77)2.67 (0.25)2.77 (0.51)Monocytes (103)0.43 (0.19)0.40 (0.07)0.44 (0.13)Cr blood (μg/L)1.14 (1.16)0.26 (0.13)0.64 (0.68)Cr urine (μg/L)1.84 (7.25)0.20 (0.14)0.68 (0.92)Cr urine (μg/g creatinine)1.20 (4.08)0.09 (0.04)0.45 (0.98)Clusterin/ApoJ (OD492)1165.1 (338.1)1140.9 (178.4)1176.3 (274.9)BMI
= body mass index; ALT (GPT) = alanine aminotrasferase; AST (GOT) = aspartate
aminotransferase; GGT =γ-glutamyltranspeptidase; LDL = low-density
lipoprotein; HDL = high-density lipoprotein; ESR = erythrocyte sedimentation
rate.Cr blood
levels in the first sampling ranged between0.1–6.1μg/L (mean 0.91; geometric mean 0.64) and urine Cr
levels ranged between 0.1–50.2μg/L  (mean 1.33; geometric
mean, 0.43) or 0.03–27.27μg/g creatinine (mean 0.87;
geometric mean 0,25). In Table 1, relative data of subgroups are presented.
ApoJ/CLU serum concentration ranged between 653 and 3075 (mean 1168; geometric
mean 1129) (OD492). None of the hematological parameters,
biochemical indicators, age, BMI, or smoking status showed any statistical
significant relation with ApoJ/CLU levels. Urine Cr levels exhibited a weak
association with ApoJ/CLU levels (r=0.160,  P=.157) raised when only SS
welders were included in the analysis but it remained insignificant (r=0.245, P=.097).
Correlation of urine Cr corrected for creatinine and ApoJ/CLU levels was also
not significant (r=0.185, P=.101) but also raised in SS welders
(r=0.217, P=.143). Having observed that trend, we analyzed data from
the first sampling concerning measurements of those welders known (from their
detailed occupational history) to be involved recently (less than 10 weeks) in
stainless steel welding. Indeed, when only this group (n=17) was included in
the analysis, the correlation coefficient (r) of ApoJ/CLU with urine Cr rose
from 0.160 to 0.333 (P = .192) and with urine Cr corrected for creatinine
rose from 0.245 to 0.453 (P = .068) but did not reach statistical significance
(P = .206).As we described previously, we implemented an intervention to lower
Cr(VI) blood levels in order to study the regulation of ApoJ/CLU levels in
human serum. All welders (n = 9) with blood Cr levels higher than 2μg/L were
assigned to two intervention groups with different grade of intensity, followed
a five-month intervention program which consisted of a significant
differentiation (lowering) in the volume and amount of stainless steel-mediated
welding. After this period, we recollected blood and urine and assayed the Cr
and ApoJ/CLU serum levels. In both groups, we found a significant reduction in
the Cr levels in all but one blood sample (Table 2). Urine levels exhibited
various trends partly explained by the intensity of intervention, in addition
to evidence that urine Cr reflects more recent exposure and it has greater
variability [26].Table 2
Chromium blood and urine levels of welders entered worksite intervention (n = 9).
WelderAgeSmoking
status1Cr
blood
(μg/L)Cr urine (μg/g creatinine)Cr
urine (μg/L)InterventionInterventionInterventionprepostprepostprepost1st58N6.14.027.2724.2750.205.202nd46H2.01.20.724.040.803.903rd38N5.00.60.640.951.500.504th40M2.90.31.030.431.100.205th36M3.70.20.310.090.900.106th37L2.52.70.652.401.201.607th42M2.11.91.131.831.202.208th35L3.01.61.942.643.103.209th35M2.71.20.204.980.303.20All mean40.783.331.5223.774.626.702.23(SD)(7.40)(1.38)(1.22)(8.83)(7.54)(16.33)(1.79)1N: nonsmokers; L: light (1–15 pack years); M: medium (16–30 pack years); H: heavy (>30 pack years)2P<.05, Wilcoxon signed ranks test.Interestingly,
we found that the reduction of Cr levels in blood due to this intervention was
related to lower ApoJ/CLU serum levels (0.77,P = .009) (Figure 1).
Higher intensity of intervention (lower exposure to hexavalent chromium) was
also related to lower ApoJ levels in a statistical significant level (1042 versus
1362, P = .032).Figure 1
Relation of ApoJ/CLU levels and the reduction of chromium blood levels (micrograms/L) (lines represent 95% mean prediction interval).The results
of multivariate analysis modeling ApoJ/CLU levels have shown that blood Cr is
the main determinant of ApoJ/CLU levels. Urine Cr holds a mild significant inverse association (negativeβ) reflecting perhaps differences in the kinetics
between compartments (Table 3).Table 3
Linear
regression results modeling for ApoJ/CLU serum levels.
VariableUnits ofββ coefficient (95% CI)P valueModelr2MODEL 10.656Cr bloodμg/L265.57 (71.38 to 1233.93).015Cr urineμg/g creatinine− 32.53 (− 70.04 to − 7.02).024MODEL 20.590pre/post interventionΔCr bloodμg/L− 122.69 (− 213.69 to  − 31.69).015


## 4. Discussion

The unifying
factor in determining toxicity and carcinogenicity for most, if not all, heavy
metals including iron, copper, chromium, vanadium, cobalt, mercury, cadmium,
and nickel is the generation of reactive oxygen and nitrogen species [27].
Metal-mediated formation of free radicals causes various modifications to DNA
bases, enhanced lipid peroxidation and altered calcium and sulfhydryl
homeostasis. Lipid peroxides, formed by the attack of radicals on
polyunsaturated fatty acid residues of phospholipids, can further react with
redox metals finally producing mutagenic and carcinogenic substances [27].We have
previously shown that welders and sandblasters, who are exposed to high levels
of heavy metals, exhibited lower ApoJ/CLU serum levels as compared to other
occupational groups [22]. This finding was unanticipated since exposure of
normal human diploid fibroblasts to low noncytotoxic levels of Cr(VI) induced
premature cellular senescence and resulted in the upregulation of the ApoJ/CLU
protein [22]. ApoJ/CLU has a nearly ubiquitous expression pattern in human
tissues and has been implicated in various physiological processes and in many
severe physiological disturbance states including ageing, cancer progression,
vascular damage, diabetes, kidney, and neuron degeneration [28]. Although
unrelated in their etiology and clinical manifestation, these diseases
represent states of increased oxidative stress [29, 30]. By combining these
findings, we proposed recently that ApoJ/CLU upregulation during ageing or at
age-related diseases does not correlate to chronological age, but it rather
relates to increased oxidative damage which can be “sensed” by the regulatory
elements of the CLU gene promoter [28].Although
chronic exposure may induce secondary molecular changes, which extend beyond
the effects of oxidative stress (e.g., inflammation or initiation of tumor
formation) [22], our current findings strengthen further the notion that the
ApoJ/CLU is a sensitive biomarker of the organismal oxidative stress. More
specifically, we report that the CLU serum levels correlate positively to the
workers exposure to heavy metals and to Cr blood and urine concentration. Cr in
blood has been shown to reflect occupational exposure to hexavalent chromium in
stainless steel welding [31] which gives support to the argument that the
significant reduction of Cr levels after the intervention is mainly due to the
reduction of hexavalent chromium which is in turn assumed to be the responsible
agent for the relation with ApoJ/CLU found in our study. However, it should be
considered that regarding the exposure to other metals there might be some
uncertainty in the assessment of the related biological effect. Lack of
detailed knowledge of the kinetics of Cr in the blood and in the elimination
compartments, especially urine, makes difficult to evaluate the correct time
and site for sampling and the number of samples that should be taken. It is
suggested that a longitudinal study in occupationally exposed participants
besides Cr and ApoJ/CLU should also include the measurement of the levels of well-known
markers of oxidative damage such as the products of lipid peroxidation
[malondialdehyde(MDA)], DNA damage (modified bases such as 8-oxo-dG), and/or
protein carbonylation in order to add firm basis to the proposed Cr-mediated
oxidative stress in the cells of occupational groups with similar exposures as
other studies have shown with various markers [32–37].In any case,
this preliminary study gives evidence that the human ApoJ/CLU gene is
responsive to the acute oxidative stress induced by heavy metals as hexavalent
chromium, and this finding may prove valuable during the monitoring and
re-evaluation of the long-term workers health effects in certain occupational
environments.Abbreviations
Apolipoprotein J/Clusterin: ApoJ/CLU, (hexavalent) chromium:
Cr(VI).



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*Source: 420578-2008-04-22.xml*