中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/26003

  

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Topic

Immunologic Effect of Workers exposed to chromium

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The objective of this study was to investigate the immunologic effect of chromic acid

exposure among electroplating workers. The study included 46 subjects selected from

five electroplating plants in central Taiwan. Each subject was interviewed using a

questionnaire and urine-chromium (Cr) concentration was assessed. Immunologic

function was evaluated by interleukin count (IL2, IL-4, IL-6, IL-8, IL-10, TNF-α, INF-γ) and levels of lymphocyte subsets (T-cell, B-cell, T4, T8, T4/T8). Results showed that IL6 and IL8 levels were significantly increased in subjects with high

urine–chromium concentration, but TNF-α levels decreased. There was no response for IL-4, IL-10 and INF. Flowcytometry was used to determine levels of lymphocyte

subsets: only B-cells percentage had a negative correlation with urine-Cr. Smoking

was an important factor that influenced levels of lymphocyte subsets. Exposure to

chromium has a detrimental effect on the immune system, so it is evident that worker

exposure to chromic acid in the electroplating workplace must be reduced to a

minimum.

Introduction

Chromates are well-known carcinogen and mutagen and are capable of inducing a

variety of DNA lesions such as strand breaks, DNA-inter-strand and DNA-protein

cross-links in both animals and cultured mammalian cells ( Cohen et al.,1993;

Mancuso 1997; Zhitkovich A et al. 1998; Werfel et al. 1998). Even before any

carcinogenic effect takes place, the immunologic consequences of hexavalent

chromium (Cr(VI))exposure have not been studies extensively. The potential

immunomudulatory effects of Cr were evaluated using a series of in vitro and in vivo

studies (Synder and Valle, 1991). Their results showed that the increased response of

cells from Cr-exposed rats indicated Cr-induced sensitization and may possibly be

used as biological markers for Cr exposure. Since Cr (VI) can easily pass the cell

membranes and it is reduced inside the cells to its trivalent form, Cr (III),

intermediates like Cr (V) and Cr (IV) and radicals are suspected to react with DNA

and cause DNA damage (Wefel et al., 1998). Moreover, Cr(VI) was also found to

induce reduction of lymphocytes in the systemic circulation. Tangawa (1991) found

that workers exposed to Cr(VI) showed a marked lymphocytopenia without alteration

of blood natural killer (NK) cells. Snyder (1996) studied IL-6 levels among

individuals in Hudson County, New Jersey (an area contaminated with chromium) and

found that IL-6 levels were significantly lower than IL-6 levels in non-contaminated

areas. Boscolo (1997) studied the effect of chromium on lymphocyte subsets and

immunoglobulins from normal population and exposed workers. The study showed

that urinary chromium had a significant positive correlation with CD16+56+NK,

CD5+CD19+ B and HLA-DR+ activated T, B and NK lymphocytes and a negative

correlation with all blood lymphocytes. Also, serum chromium was significantly

correlated with all blood lymphocytes and HLA-DR+, CD3-HLA-DR+ and

indicators fluctuate a great deal after initial exposure to a toxic substance and this

makes it very difficult to obtain consistent data. Previous studies by current authors

showed that chromium electroplating plants in central Taiwan and found that workers

there had a variety of severe health problems such as renal dysfunction (Liu et al,

1998) respiratory problems (Kuo et al., 1997) and increased sister chromatid exchange

(Lai et al, 1998) as a result of exposure to Cr. There is no data available in Taiwan

concerning the effect of chromium on the immune system. The objective of this study

is to investigate to what extent chromium effects immunologic function. Also, this

study can be used to create a database of long term exposure to chromium, including

health data and environmental monitoring. This study will be used as a reference by

the Taiwan government and electroplating plants, for the purposes of improving safety

conditions in the electroplating workplace.

Materials and Methods

Subjects: The 46 subjects were selected from five Cr electroplating plants in central

Taiwan. None of the subjects had had any immunologic disease, prescription medicine

pertaining to immune function or organ transplant one month prior to the

commencement of the study.

Methods: Each worker was interviewed by questionnaire. Basic data, workplace

conditions and health status were determined. A urine sample was taken from each

subject at the end of a shift and was analyzed for chromium by atomic absorption

spectrophotometry (AAS) within one week. Detailed information has been outlined in

a previous study (Kuo, 1997). The data was divided into three groups based on the

concentration of chromium in urine: (1) Low urine-Cr, less than 25% (<1.13ug/g cre)

(2) Intermediate urine-Cr, 25-75% (1.13-6.41ug/g cre) (3) High urine-Cr, over 75%

Immunologic function test

Enzyme-linked immunosorbent assay (ELISA) was used to determine the

interleukin-2, 4, IL-6, IL-8, IL-10, TNF-α and INF-γ. The standard procedure for determining interleukins (as recommended by R&D systems) was followed. The

sandwich immunoassay technique was used. Blood samples were added to the

interleukin microtiter plate and a color reagent was then added. Within 30 minutes, an

ELISA reader analyzed the samples. To establish a calibration curve, five

concentrations of the interleukin standard were prepared. Each sample was duplicated

and the coefficient of variation (CV) was less than 10%.

All data were analyzed using SAS/pc+ 6.04 statistical software (SAS/STAT 1986).

Analysis methods included analysis of variance (ANOVA), correlation analysis and

the general linear model.

Results

Table 1 shows three groups divided according to urine-Cr and immune function.

Interleukin-2, IL-4, IL-10 and IFN were not detected in any workers urine samples.

However, IL-6, IL-8 and TNF-α were all present. IL-6 and IL-8 levels increased in direct proportion to urine-Cr concentration, but only IL-8 had a statistical significance

(High urine-Cr group = 64.08. Low urine-Cr group = 23.57). There was a negative

correlation between TNF-α and urine-Cr, but there was no statistical significance. There was a negative correlation between urine-Cr and T-cell and B-cell. However,

only B-cell had a significant difference (High urine-Cr group = 8.80%. Low urine-Cr

group = 12.89%). The T4/ T8 ratio was correlated positively with urine-Cr. (High

urine-Cr group = 1.87. Low urine-Cr group = 1.26). The p value however is 0.074.

There was no significant difference in the other indicators: T4, T8, lymphocyte,

Table 2 shows the correlation of urine-Cr, airborne Cr(VI) and work duration

with immune indicators. There is a positive correlation of urine-Cr with IL-6 and IL-8

(only IL-6 was significant). TNF-α and B-cell correlated negatively with urine-Cr (r = -0.19 and –0.24, respectively). T4/ T8 ratio correlated positively with urine-Cr (r =

0.27). Only monocyte correlated positively with airborne Cr(VI) (r = 0.28). Work

duration did not correlate with any of the immune indicators.

Table 3 shows the factors that influence the immune indicators using multiple

regression. The high urine group compared to the low group for IL-6 and IL-8 has a

statistical significance. However, there was no significance for gender, age and

smoking. No factors explained levels of TNF-α; there was a slight negative correlation with urine-Cr. After adjustment of the other factors, the results showed that

the high urine-Cr group and the medium urine-Cr group compared to the low urine-Cr

group for B-cell both had negative statistical difference. Smokers had higher T4

levels, granulocyte and T4/T8 ratios than non-smokers. However, lymphocyte levels

were lower in smokers than in non-smokers. Males had lower T-cell levels than

females. Age and electroplating work did not affect immune function.

Discussion

Most electroplating factories in Taiwan are small-scale and there is a lack of provision

of personal protective equipment for workers. Previous studies (Kuo et al., 1997;

Liu et al., 1998; Lai et al., 1998) have shown that in hard Cr electroplating factories,

the absence of adequate safety procedures and the requirement of workers to handle

airborne Cr.), have resulted in the development of serious health problems among

workers. Concentration of airborne Cr(VI) in electroplating factories varies greatly.

For example, near the electroplating tank Cr(VI) is highest whereas in the

administrative office levels of Cr(VI) are very low. There is no existing data in

Taiwan to show the effect of chromium on the immune function of electroplating

workers. Glaser (1985) reported that respiratory defense and immunologic functions

were stimulated or inhibited depending on dose and time of Cr(VI) inhalation. The

humoral immune system was still stimulated at sub-chronic low chromium aerosol

concentration of 100mg/m3, but significant depressed at 200 mg/m3. Boscolo (1997)

studied the effects of chromium on lymphocyte subsets and immunoglobulins (Ig)

from normal population and exposed workers. The results showed that in the workers’

blood, CD₄+ helper-inducer, CD₅-CD₁₉+B, CD₃-CD₂₅+ activated B and

CD₃-HLA-DR+ activated B and natural killer (NK) lymphocytes were significantly

reduced (about 30-50%). Boscolo suggests that Cr(III) may be involved in the

mechanisms regulating the immune response in humans. Tanigawa (1991) also

reported a decrease in Leu-11a negative lymphocytes in relation to NK cell activity in

chromate workers. The lymphocyte subpopulation may provide a useful indicator of

the effects of exposure to chromium. The current study measured T-cell and B-cell

immunology tests. Various interleukin levels were also assessed. Interleukins may be

described as lymphocyte-activating factors that stimulate the proliferation of murine

thymocytes. The above-mentioned immunologic indicators can be used to evaluate the

effect of short-term or long-term exposure of electroplating workers to chromium.

It is interesting to note that exposure to chromium had no effect on the levels of

IL-2, IL-4, IL-10 and IFN. However, IL-6, IL-8 and TNF were detected in workers’

blood samples. Urine-Cr levels correlated positively with IL-6 and IL-8 levels. This

data is consistent with Snyder’s (1986) study of IL-6 levels among individuals in

Hudson County, New Jersey (an area contaminated with chromium). IL-6 works in

conjunction with T helper cells and can stimulate the production of antibodies.

Therefore, a decrease in IL-6 levels will result in decreased antibody production,

which in turn will reduce immune function. Very probably the ability to promote

differentiation of B lymphocytes into antibody-secreting plasma cells, and the

induction of acute-phase protein synthesis in the liver in response to environmental

stresses. In addition, IL6 can act as enhancing signal for various T lymphocyte

activities (IL2 production and cell proliferation) and also induce a febrile response in

vivo. However, IL-8 plays an important role in the inducing acute or chronic

inflammatory response. IL-8 stimulates neutrophil, basophil and T-lymphocytes to

cells, macrophages, neutrophils and mast cells. T lymphocytes join in late when

foreign antigens are presented to initiate immune responses. These effects are

achieved partly by release at the site of injury of chemoattractant agent (such as IL 8).

Chromium is the materials most oftens used for joint implantation. Therefore, Wang’s

study was aimed at investigateing the cytotoxicity of Cr and weather Cr affects T and

B cell profiferation and release of cytokines by human peripheral blood mononuclear

cells (PBMC) in vitro. The results shed light on how Cr impaired immune response

and cytokines release, suggesting that patients with an extensive exposure to

chromium may develop immune dysfunctions. Because cytokines are

immunoregulatory molecules, primarily synthesized by leukocytes, they play an

extremely important role in the communication network that links inducer and

effector immune cells, producing effective resistance to infection. So our results have

showed that workers with an extensive exposure to Cr may develop immune

dysfunction. Consequently, a long-term exposure of workers to Cr may lead to

substantial defects in immune functions including disruption of lymphocyte and

macrophage differentiation in alveolar macrophage (AM). AMs are important

phagocytic and secretary cells that participate in various complex immunologic and

inflammatory processes. Gao(1992) have shown that Cr(VI) is able to induce DNA

vivo. Furthermore, the formation of the oxidized deoxynucleoside

8-hydroxydeoxyguanosine (8-OhdG) in isolated DNA after treatment with Cr(VI) or

Cr(V) compounds suggested that the mechanism of toxicity might involve the

References:

Boscolo P, Di Gioacchino M, Bavazzano P, White M, Sabbion E (1997) Effects of chromium on lymphocyte subsets an immunoglobulins from normal population and exposed workers. Life Sci 60:1319-25.

Cohen MD, Kargacin B, Klein CB, Costa M (1993) Mechanisms of chromium carcinogenicity and toxicity. Crit Reviews in Toxicol 23:255-81.

Deforg LE, Preston AM, Takenchi E, Kenney J, Boxer LA, Remick DG (1993) Regulation of interleukin 8 gene expression by oxidant stress. J Bio Chem 268:25568-76.

DeForge LE, Fantone JC, Kenney JS, Remick DG (1992) Oxygen radical scarverigers selectively inhibit interleukin 8 production in human whole blood. J Clin Invest 90:2123-9.

Faux SP, Gao M, Chipman JK, Levy LS (1992) Production of 8-hydroxydeoxyguanosin in isolated DNA by chromium and chromium (V). Carcinogenese 13:1007-9.

Gao M, Binks SP, Chipman JK, Levy LS, Braithwaite RA, Brown SS (1992) The in-vitro induction of DNA single strand breaks in human lymphocytes by chromium compounds. Human Exp Toxciol 11:77-82.

Glaser U, Hochrainer D, Kloppel H, Kuknen H (1985) Low levels chromium (IV) inhalation effects on alveolar macrophage and immune functions in Wistar rats. Arch Toxciol 57:280-6.

Kuo HW, Lai JS, Liu TI (1997) nasal septum lesions and lung function in workers exposed to chromic acid in electroplating factories. Int Arch Occup Environ Health 58:29-32.

Lai JS, Kuo HW, Liao FC, Lien CH. Sister chromatid exchange induced chromium compounds in human lymphocyte. Int Arch Occup Environ Health 71:550-3. Liu CS, Kuo HW, Lai JS, Lin TI (1998) Urinary N-acety-B-glucosaminidase as

indicator of renal dysfunction in electroplating workers. Int Arch Occup environ Health 1998:62:452-7.

Mancuso TF (1997) Chromium as an industrial carcinogen. Part I. Am J Ind Med 31:129-39.

Martin F, Santolaria F, Batista N et al. (1999) Cytekine levels (IL6 and IFN-γ) acute phase response and nutritional status as prognostic factors in lung cancer.

Cytokine 11:80-6.

SAS/STAT (1986) User’s guide, release 6.04. SAS, Cary, North Carolina.

Sugiyama M (1992) Role of physiological antioxidants in chromium(VI)-induced cellular injury. Free radical Biological Med 12:397-407.

Syder CA, Valle CD (1991) Immune function assays as indicators of chromate exposure. Environ Health Perspect 92:83-6.

Synder CA, Udasin I, Waterman SJ et al. (1996) reduced IL-6 levels among individuals in Hudson county, New Jersey, an area with contaminated with chromium. Arch Environ Health 51:26-28.

Tangawa T, Araki S, Ataki T, Mitato N (1991) A decrease in Leu 11 a negative lymphocytes in relation to natural killer cell activity in chromate workers. Br J Ind Med 48:211-3.

Wang JY, Tsukayama DJ, Wicklund BH, Gustilo RB (1996) Inhibition of T an B cell proliferation by titanium, cobalt, and chromium: role of IL2 and IL6. J Biolmed Materials Res 32:655-61.

Table 1. Comparison of immune function based on urinary Cr concentration Low urine-Cr group Moderate urine-Cr group High urine-Cr group P value IL-6 0.65±0.55 a 0.84±0.60 1.18±0.71 NS IL-8 _{23.57±50.16 37.15±45.4 64.08±35.22 <0.01} TNF-α 2.90±4.51 1.98±1.82 1.90±2.09 NS T4 34.35±6.91 36.04±7.83 35.40±7.66 NS T8 _{28.26±6.59 28.04±7.33 21.6±6.26 Ns} T-cell _{66.31±9.72 66.40±10.93 56.6±9.96 NS} B-cell _{12.89±3.44 9.90±4.18 8.80±3.42 <0.05} Lymphocyte 29.21±8.07 25.04±9.65 28.4±9.71 NS Monocyte 4.94±1.50 5.31±3.06 4.60±1.81 NS Granucyte _{65.84±8.83 69.72±10.26 66.8±11.2 NS} T₄/T₈ ratio _{1.26±0.33 1.28±0.52 1.87±1.07 NS} a _means±SD

Low urine-Cr group
urinary Cr concentration <1.13(µg/g Cre)

Moderate urine-Cr group
urinary Cr concentration
1.136.41(µg/g Cre) High urine-Cr group
urinary Cr concentration >6.41(µg/g Cre)

Table 2. Correlation between urinary and airborne Cr concentrations, work duration and immune function

Urinary Cr conc. (g/g cre) Airborne Cr conc. (mg/m3) Work duration (month) IL-6 0.35** -0.004 -0.06 IL-8 0.22 0.13 -0.12 TNF-α -0.19 -0.12 -0.11 T4 0.05 -0.06 0.07 T8 -0.22 -0.08 0.20 T-cell -0.18 -0.008 0.19 B-cell -0.24* 0.05 0.03 Lymphocyte -0.002 0.11 0.07 Monocyte -0.05 0.28* 0.04 Granucyte 0.01 -0.16 -0.07 T₄/T₈ ratio 0.27 -0.01 -0.06 * p<0.1 **p<0.05

Table 3. Analysis of risk factors for immune function using a multiple regression model

IL-6 IL-8 _TNF- Variables B(SE) a B(SE) B(SE)

Urine-Cr

Mediate group(Low group=1) 0.38(0.26) 16.24(19.50) -0.63(1.30) High group(Low group=1) 0.69(0.26)** 38.74(20.10)* -0.85(1.34)

Gender(Male=1) -0.05(0.29) 20.17(21.78) 1.41(1.45) Age (years) 0.01(0.01) 0.98(0.70) -0.01(0.04) Electroplating task(no=1) 0.01(0.01) -0.36(1.26) -0.04(0.08) Smoking habit(no=1) 0.05(0.25) 8.41(18.94) 0.77(1.26) R2 0.28 0.24 0.14 a_{B(SE) * p<0.1 **p <0.05 **p <0.01}

Table 3. Analysis of risk factors for immune function using a multiple regression model (continue)

T4 T8 T cell B cell Lympho Mono Grannel T4/T8 ratio variable B(S.E)a B(S.E)a B(S.E)a B(S.E)a B(S.E)a B(S.E)a B(S.E)a B(S.E)a Urine-Cr Moderate group (low group=0) -0.03 (2.54) -1.78 (2.28) -7.81 (8.55) -2.87** (1.41) -1.10 (2.93) 0.94 (0.90) 0.16 (3.18) 0.07 (0.19) High group (low group=0) -0.23 (4.00) -6.49 (3.59) -8.82 * (4.93) -4.29* (2.23) -2.48 (4.63) -0.67 (1.42) 3.03 (5.02) 0.53* (0.30) Gender (male=0) 3.26 (3.75) -0.78 (3.37) 1.07 (4.62) 2.45 (2.09) 1.08 (4.34) 0.71 (1.33) -1.73 (4.70) 0.24 (0.28) Age (years) 0.19 (0.11) 0.22 ** (0.10) 0.36* (0.14) -0.01 (0.06) -0.20 (0.13) -0.06 (0.04) 0.26 (0.14) -0.01 (0.01) Electroplating task (no=1) -0.41 ** (0.20) -0.49** (0.18) -1.02** (0.25) 0.01 (0.11) 0.45* (0.23) 0.08 (0.07) -0.53** (0.25) 0.01 (0.01) Smoking habit (no=1) 3.84 (3.05) -5.29 (2.74) -4.22 (3.75) 0.91 (1.70) -4.52 (3.52) 0.56 (1.08) 3.86 (3.82) 0.45 ** (0.23) R2 0.22 0.33 0.43 0.22 0.32 0.07 0.31 0.21 a _{B(SE) * p<0.1 **p <0.05 **p <0.01}