KEGIATAN ILMIAH I

BIOMONITORING OF VINYL CHLORIDE EXPOSURE

ABBAS ZAVEY NURDIN

1206236331

Pembimbing:

Dr. dr. Astrid W. Sulistomo, MPH, SpOk

FAKULTAS KEDOKTERAN
PROGRAM PENDIDIKAN DOKTER SPESIALIS
KEDOKTERAN OKUPASI
JAKARTA
2013
 CHAPTER I
INTRODUCTION

Vinyl Chloride (VC) is organochloride

VINYL CHLORIDE
with the formula H2C:CHCl or called Vinyl
chloride monomer (VCM), or Kloroetena. Vinyl
Chloride is important industrial chemical
substance mainly used to produce the polymer
polyvinyl chloride (PVC), which is a popular
ingredient as frame windows and doors, clothing, cable IUPAC NAME: Chloroethene
insulation, and piping. Characteristics of VC will be
OTHER NAME: Vinyl chloride
described by International Programme On monomer VCM, Chloroethylene
Chemical Safety below:
Molecular
C2H3Cl
formula
Identity, physical and chemical properties
“Under ambient conditions, VC is a Molar mass 62.50 g mol−1
colourless, flammable gas with a slightly sweet
odour. It has a high vapour pressure, a high value Appearence Gas, tidak berwarna
for Henry's Law constant and a relatively low
water solubility. It is heavier than air and is soluble Density 0.911 g/ml
in almost all organic solvents. It is transported in
liquid form under pressure. At ambient Melting point -153.8 °C, 119 K, -
245 °F
temperatures in the absence of air, dry purified VC
is highly stable and non-corrosive but above Boiling point -13.4 °C, 260 K,
8 °F
450°C, or in the presence of sodium or potassium
hydroxide, partial decomposition can occur. Sulubility in water 2.7 g/L (0.0432
mol/L)
Combustion of VC in air produces carbon dioxide
and hydrogen chloride. With air and oxygen, very Flash point -61 ° C (-78 ° F)
explosive peroxides can be formed, necessitating a
continuous monitoring and limitation of the oxygen content, particularly in VC recovery
plants. In the presence of water, hydrochloric acid is formed.
The concentration of VC in air can be monitored by trapping it on adsorbents and,
after liquid or thermal desorption, analysis by gas chromatography. In ambient air
measurements, several adsorbents in series or refrigerated traps may be needed to increase the
efficiency of trapping. Peak concentrations at workplaces can be measured with direct-
reading instruments based on, for instance, Flame ionization detector/FID or Photoionization
detector/PID. In continuous monitoring, Infrared/IR and Gas chromatography (GC)/FID
analysers combined with data logging and processing have been used. In analysis of VC in
liquids and solids, direct injection, extraction and more increasingly head space or purge-and-
trap techniques are applied. Also in these samples, VC is analysed by GC fitted to, for
instance, FID or Mass Spectrometry/MS detector. Vinyl Chloride is not known to occur
naturally although it has been found in landfill gas and groundwater as a degradation product
of chlorinated hydrocarbons deposited as solvent wastes in landfills or in the environment of
workplaces using such solvents. Vinyl Chloride is also present in cigarette smoke.

Environmental transport, distribution and transformation

Owing to its high vapour pressure, VC released to the atmosphere is expected to exist
almost entirely in the vapour phase. There are indications for wet deposition. Vinyl Chloride
has a relatively low solubility in water and has a low adsorption capacity to particulate matter
and sediment. Volatilization of VC is the most rapid process for removal of VC introduced
into surface waters. Half-lives reported for volatilization from surface waters range from
about 1 to 40 h.

Environmental levels and human exposure

There is very little exposure of the general population to VC. Atmospheric
concentrations of VC in ambient air are low, usually less than 3 µg/m3. Exposure of the
general population may be higher in situations where large amounts of VC are accidentally
released to the environment, such as in a spill during transportation. However, such exposure
is likely to be transient.
The main route of occupational exposure is via inhalation and occurs primarily in
VC/PVC plants. Vinyl Chloride has occasionally been detected in surface waters, sediment
and sewage sludges, with maxima of 570 µg/litre, 580 µg/kg, and 62 000 µg/litre,
respectively. Maximal VC concentrations in groundwater or leachate from areas
contaminated with chlorinated hydrocarbons amounted to 60 000 µg/litre (or more). High
concentrations (up to 200 mg/litre) were detected in well water in the vicinity of a PVC plant
10 years after leakages. The few data available show that VC can be present in tissues of
small aquatic invertebrates and fish. Some recent studies have identified VC in PVC-bottled
drinking-water at levels below 1 µg/litre. The frequency of occurrence of VC in such water is
expected to be higher than in tap water. Packaging with certain PVC materials can result in
VC contamination of foodstuff, pharmaceutical or cosmetic products, including liquors (up to
20 mg/kg), vegetable oils (up to 18 mg/kg), vinegars (up to 9.8 mg/kg) and mouthwashes (up
to 7.9 mg/kg).” 7
According to the regulation in Indonesia through PERMENAKERTRANS NOMOR
PER.13/MEN/X/2011 about Threshold Limited Value of chemical and physical factors in
the workplace, it is explained that vinyl chloride is chemical substance with Threshold
Limited Value 5 ppm or 13 mg/m3 with molar mass 62,50 g mol−1 .8
 CHAPTER II

LITERATUR REVIEW

REVIEW
7 Journal articles have been reviewed :

1 Urinary thiodiglycolic acid levels for vinyl chloride monomer-exposed polyvinyl

chloride workers by TJ Cheng et al: Thiodiglycolic acid (TdGA) is the major
metabolite of vinyl chloride monomer (VCM) detected in human urine. Although urinary
TdGA has been reported to be associated with ambient VCM exposure, the relationship
between urinary TdGA and a low level of air VCM is not clear. Questionnaires were
administered to 16 polyvinyl chloride manufacturing workers to obtain a detailed history
of occupation and lifestyle. For each worker, personal air monitoring for VCM was
performed and a time-weighted average for VCM exposure was calculated. The urinary
TdGA levels at the end of a work shift, and at the commencement of the next shift, were
also assessed for each worker. Urine analysis revealed that TdGA levels at the beginning
of the next shift were higher than those at the end of that shift. Workers experiencing a
VCM exposure greater than 5 ppm in air revealed a urinary TdGA level significantly
greater than those experiencing a VCM exposure of less than 5 ppm (P < 0.05). The best
fit of regression for urinary TdGA on air VCM was Y = 1.06 + 0.57X for urine collected
at the commencement of the following work shift, where X is the air VCM concentration
and Y is the urinary TdGA concentration (r2 = 0.65, P < 0.01). We conclude that the
urinary TdGA level is best detected at the commencement of the next shift and that it can
be used as an exposure marker for polyvinyl chloride workers when the air VCM level to
which they are exposed is greater than 5 ppm.1

2 The effect of genetic polymorphisms in the vinyl chloride metabolic pathway on

mutagenic risk. Jennifer Schindler et al: Vinyl chloride (VC) is a human carcinogen
known to undergo metabolism by cytochrome P450 2E1 (CYP2E1) to reactive
intermediates that can cause oncogene and tumor suppressor gene mutations and that are
further metabolized by acetaldehyde dehydrogenase (ALDH2) and glutathione-S-
transferases (GSTs) to nonmutagenic end products. These metabolic enzymes have
known polymorphisms that could lead to increased levels of the VC reactive
intermediates and thus an increased risk for mutations and cancer following exposure.
Using restriction fragment length polymorphism (RFLP) analysis, we have examined a
 cohort of 597 French VC orkers for polymorphisms in CYP2E1, ALDH2, GSTM1 and
GSTT1 in relation to the occurrence of mutant oncogene and tumor suppressor gene
biomarkers that are attributable to VC exposure. The presence of the biomarkers for
mutant rasp21 and mutant p53 was found to be highly significantly associated with
cumulative VC exposure (P for trend <0.0001). The presence of the CYP2E1 variant c2
allele was found to be significantly associated with the presence of either or both mutant
biomarkers even after controlling for potential confounders including cumulative VC
exposure (OR = 2.3, 95% CI = 1.2–4.1), and the effects of the c2 allele and VC exposure
were approximately additive. GSTT1 null status was found to have an increased, but not
significant association with the presence of either or both biomarkers after controlling for
confounders (OR = 1.3, 95% CI = 0.8–2.0). These results suggest the existence of a
possible gene–environment interaction between polymorphisms in the VC metabolic
pathway and VC exposure that could contribute to the variable susceptibility to the
mutagenic effects of VC in exposed populations. 2

3 Interactions of chemical carcinogens and genetic variation in hepatocellular

carcinoma, Jing Zhang Yu: Vinyl chloride is primarily metabolized in the liver by the
CYP2E1 to form the electrophilic metabolites chloroethylene oxide and
chloroacetaldehyde[90]. These metabolites are thought to be the reactive intermediates
involved in the formation of VC-DNA adducts. The promutagenic properties of these
adducts have been characterized extensively in vivo and in vitro and involve mainly base
pair substitution mutations[91]. Metabolism of the reactive intermediates is thought to
involve several pathways that rely on CYP2E1, aldehyde dehydrogenase 2, GSTs,
microsomal epoxide hydrolase and other enzymes, presumably to generate less reactive
metabolites for excretion[92]. All of those enzymes are known to have polymorphic
variants with altered activities that could produce variable VC metabolism[93]. Such
variable metabolism could account for differing susceptibilities to the carcinogenic effects
of VC in exposed individuals. The GST family is known to be involved in the metabolism
of environmental chemical carcinogens including vinyl chloride monomer; it plays
critical roles in protection against products of oxidative stress and electrophilic
compounds[94,95]. So far, no direct evidence has shown that genetic polymorphisms of
metabolizing enzymes are correlated. 3

4 Evaluation of liver enzyme levels in workers exposed to vinyl chloride vapors in a

petrochemical complex: a cross-sectional study by Attarchi Mir Saeed et al: In this
 study, liver enzyme levels of 52 workers were compared to 48 control workers using the
T-test. The cases all worked in a PVC production unit in a petrochemical complex and the
controls were randomly selected from office personnel of the same complex. A
questionnaire was also filled in about information such as age, weight, work history, etc.
in both groups. Result mean comparisons for ALP and GGT using T-test showed
statistically significant differences between the two groups. For AST, ALT and bilirubin
(total, direct) the mean was higher in the case group but this difference was not
statistically significant. This study showed that mild exposure to VCM can cause mild
liver cholestasis. So, using cholestasis assessment tests such as ALP and GGT should be
considered in periodic assessment of liver function in PVC producing units. 4

5 Immunoquantitation of von Willebrand factor (factor VIII - relatedantigen)

in vinyl chlorideexposed workers, Fromen O, et al: Angiosarcoma of the liver is a rare
malignant tumor which has been associated with occupational exposure to vinyl chloride
(VC). We have determined by ELISA the level of von Willebrand factor (vWf) in the
serums of 107 VC-exposed workers, active or retired, and of 133 blood donors used as
controls. The vWf level was slightly but significantly higher in the VC-exposed group
than in the control group (P = 0.035). Seventeen VC-exposed workers exhibited a raised
level of vWf, with no biochemical sign of hepatic disturbance, nor any evidence of
illness; only one of them exhibited elevated alkaline phosphatase and gamma-glutamyl
transpeptidase values. The vWf serum level of 3 patients with hepatic angiosarcoma
associated to VC-exposure was markedly elevated. These increased levels of vWf in VC-
exposed workers most likely reflect an increased activity of liver endothelial cells;
whether an elevated level of vWf could be associated with increased risk of developing
liver angiosarcoma remains to be determined. 5

6 Messenger RNA expression and genetic polymorphisms of cell cycle control genes
and chromosomal aberrations in Chinese vinyl chloride monomer-exposed workers.
Y.L, Qiu et al: p53 mRNA expression of VCM-low- and high-exposure groups was
significantly lower than that of nonexposed group (P < 0.001), but p21 mRNA expression
of the two VCM-exposed groups was significantly higher than that of the nonexposed
group (P < 0.001). This study did not find the relationship between chromosomal
aberrations, genotypes, and the expression of p53, p21, and CCND1. Conclution:
Messenger RNA expressions of p53 and p21 are changed with VCM-exposure status.6
 CHAPTER III
DISCUSSION

International Programme On Chemical Safety describe that “Vinyl Chloride is a

gas so the most significant exposures are respiratory. Following inhalation exposure,
absorption is rapid in humans and most subsequent metabolism occurs in the liver. Phase
I metabolism is primarily via the cytochrome P-450 isoenzyme 2E1 (CYP2E1) to
generate the reactive intermediates chloroethylene oxide (CEO) and chloracetaldehyde
(CAA) which are further metabolized in Phase II reactions by glutathione-S-transferases
(GSTs) and aldehyde dehydrogenase 2 (ALDH2) to end-products for ultimate excretion.
However, CEO and CAA can readily interact with cellular macromolecules, including
DNA, to produce promutagenic effects. Because CEO tends to react much more rapidly
with nucleic acids than CAA, it is usually regarded as the most relevant electrophile for
the generation of DNA adducts and consequent mutagenic effects; the greater biological
relevance of CEO is also supported by comparisons of the adduct profile of VC with that
of 2,2-dichloroethyl ether, which only produces CAA as a metabolite.VC
biotransformation to CEO probably occurs principally in hepatocytes, but the epoxide can
also reach and react with adjacent sinusoidal lining cells, so that mutagenic effects can
occur in parenchymal liver cells and non-parenchymal endothelial cells, providing a
logical rationale for the association between VC exposure. The major VC-associated liver
DNA adduct is 7-(2-oxoethyl)guanine, comprising up to 98% of all adducts formed.
However, this adduct is eliminated from the DNA with a very short half-life, principally
by chemical depurination, and is not considered to be promutagenic. On the other hand,
three etheno DNA adducts are also formed in much less abundance, but they are known to
be promutagenic. These are: N 2 ,3-ethenoguanine (εG); 1,N 6 -ethenoadenine (εA); and
3,N 4 -ethenocytosine (εC). It should be noted that these etheno-DNA adducts can also be
found in tissues from unexposed humans and animals because they can be produced
endogenously through the interaction of lipid peroxidation-derived aldehydes and
hydroxyalkenals. However, VC exposure can increase the level of these adducts 10-100
fold over background in the hepatocytes and non-parenchymal liver cells of exposed
animals. There are several potential mechanisms by which the VC-induced adducts could
be repaired before they have a chance to cause mutations. As noted above, the oxoethyl
adduct is removed rapidly by chemical depurination. The potential repair of the etheno
adducts is more complicated. The 1,N 6 -εA adducts are recognized and removed by 3-
methyl adenine DNA glycosylase which is part of the Base Excision Repair (BER)
pathway; this is accomplished by hydrolyzing the N-glycosidic bond between the
damaged base and deoxyribose, leaving an abasic site in the DNA. The BER apparatus
includes numerous other proteins that complete the repair at the abasic site once the
adduct is removed
After inhalative or oral exposure to low doses, VC is metabolically eliminated
and non-volatile metabolites are excreted mainly in the urine. Comparative investigations
of VC uptake via inhalation revealed a lower velocity of metabolic elimination in humans
than in laboratory animals, on a body weight basis. However, when corrected on a body
surface area basis, the metabolic clearance of VC in humans becomes comparable to that
of other mammalian species. With increasing oral or inhalative exposure, the major route
of excretion in animals is exhalation of unchanged VC, indicating saturation of metabolic
pathways.
The promutagenic properties of etheno-DNA adducts that are not fully repaired
by one or another of the DNA repair pathways have been well-documented in
experimental systems in vitro, as well as in vivo in bacterial and mammalian cells. The εG
adduct generates G->A base changes; the εA adduct generates A->G, A->T and A->C
base changes; and the εC adduct generates C->A and C->T base changes. These
experimental results are consistent with the tumor mutational spectra identified in
exposed animals and humans in oncogenes and tumor suppressor genes, although there
are both important similarities and differences between the patterns seen in the animal
tumors and the human tumors. Both Hepatocellular carcinoma/ HCCs and Angiosarcoma
of the livers/ASLs in VC-exposed rats were found to have mainly A->T transversions in
the cancer-related H-ras and p53 genes. For example, seven of eight VC-induced HCCs
in VC-exposed rats had an A->T transversion at Codon 61 of H-ras; 11 of 25 ASLs and
one of eight HCCs in VC-exposed rats had mutations in p53, all but one of which were
base pair substitutions with nine at A:T base pairs (5 A->T, 2 A->G, 2 A->C) and three at
G:C base pairs (all G->A), and two of the A->T transversions occurred at the same site
(the first nucleotide of Codon 253). On the other hand, ASLs in VC-exposed humans
have been found to have exclusively A->T transversions in p53 (three of six, including
one at the first nucleotide of Codon 255 which corresponds to the same mutation in
Codon 253 seen in rats). Of note, studies of p53 mutations in 21 human ASLs that were
not associated with VC exposure, only two had mutations , neither of which were A->T
transversions. Studies of VC-associated HCCs have also found frequent p53mutations,
occurring in 11 of 18 cases, although only two of these were A->T transversions. Both
ASLs and HCCs in VC-exposed humans have been found to have primarily G->A
transitions in the K-ras oncogene at Codons 12 and 13. For example, in studies of VC-
associated HCCs, 5 of 12 tumors were found to have K-ras mutations at Codons 12 and
13, three of which were G->A transitions. Even more striking, in studies of K-ras gene
mutations in VC-associated ASLs, G->A transitions were found in 23 of 33 tumors with
the vast majority of these (17 of 23) occurring at Codon 13. Again of note, studies of K-
ras mutations in 24 ASLs that were not associated with VC exposure did find G->A
transitions in seven cases, but all of them were at Codon 12, indicating that the Codon 13
mutation may be relatively specific for VC-induced ASLs. Interestingly, in studies of
both ASLs and HCCs in VC-exposed humans, five cases were also found to contain K-
ras mutations in the histologically normal adjacent tissue, three of which were G->A
transitions and two of which occurred at Codon 13; likewise, at least one case of a non-
dysplastic pre-angiosarcomatous liver lesion has been found to contain the characteristic
G->A transition at Codon 13 of K-ras. These results suggest that these VC-associated
mutations, particularly the Codon 13 K-ras mutation, may be a relatively early event in
VC carcinogenesis, and thus the occurrence of these mutations may be useful biomarkers
of cancer risk in exposed individuals.
The G->A transition at Codon 13 of K-ras results in the substitution of an
aspartic acid (Asp) for the normal glycine (Gly) at amino acid residue 13 in the encoded
p21 protein product. This substitution is believed to be oncogenic, having been identified
in other human tumors as well. The oncogenic mechanism of action of this substitution is
thought to be through the production of a conformational change in p21 which may be
responsible for altering its intrinsic GTPase activity, thus affecting signal transduction
within the cell leading to uncontrolled growth and division. Similarly, the A->T
transversions at various codons of p53 produce their corresponding amino acid
substitutions in the encoded p53 protein product, all changes that have been shown to
cause the protein to adopt its so-called "malignant" conformation with a concomitant loss
of its normal tumor suppressor activity. These protein changes provide a useful indicator
of the pathogenic consequences of the occurrence of the corresponding mutations as well
as convenient intermediate biomarkers of the VC effect to study the molecular
epidemiology of VC carcinogenesis in exposed human populations. Point mutations have
been detected in p53 and ras genes in tumours from highly exposed (before 1974)
autoclave workers with liver angiosarcoma (ASL) and another VC worker with
hepatocellular carcinoma.
Biological markers that have been investigated as indicators for VC exposure or
VC-induced effects include:
1. Excretion of VC metabolites (e.g., thiodiglycolic acid),
2. Genetic assays (e.g., chromosomal abnormalities or micronucleus assay),
3. Levels of enzymes (e.g., in liver function tests),
4. Von Willebrand factor: The immunoquantitation of the level of the von Willebrand
factor (vWf; factor VIII-related antigen, a large multimeric plasma glycoprotein in the
blood clotting system) using an enzyme-linked immunosorbent assay (ELISA)
5. Serum oncoproteins (p21 and p53) and/or their antibodies as biomarkers of VC-
induced effects.”7
The biological markers examination should ideally be examined as a whole in
workers exposed to vinyl chloride, but if it is limited in terms of budget, time checks and
spot checks may not get in Indonesia, my opinion on a general check up periodically for
examination excretion of VC metabolites to determine the possibility of exposure into the
body is by finding thiodiglycolic acid in urine and examined levels of enzymes in liver
function tests such as AST, ALT and bilirubin to know early signs of liver dysfunction
and an impaired hepatic function obtained the workforce immediately removed from the
exposure and examined for signs of enlarged liver or hepatomegaly, if the worker earned
magnification examination should be performed.
 CHAPTER IV
SUMMARY

Vinyl Chloride (VC) is organochloride and important industrial chemical substance

mainly used to produce the polymer polyvinyl chloride (PVC), which is a popular ingredient
as frame windows and doors, clothing, cable insulation, and piping. The main route of
metabolism of VC after inhalation or oral uptake can cause a specific pathological syndrome
found in VC workers called the "vinyl chloride illness". Symptoms described were earache
and headache, dizziness, unclear vision, fatigue and lack of appetite, nausea, sleeplessness,
breathlessness, stomachache, pain in the liver/spleen area, pain and tingling sensation in the
arms/legs, cold sensation at the extremities, loss of libido and weight loss.

The study of biological markers dercribe that vinyl chloride biomonitoring marker are
Excretion of VC metabolites (e.g., thiodiglycolic acid), genetic assays (e.g., chromosomal
abnormalities or micronucleus assay), levels of enzymes (e.g., in liver function tests), von
Willebrand factor, and serum oncoproteins (p21 and p53) and/or their antibodies as
biomarkers of VC-induced effects.

The biological markers examination should ideally be examined as a whole in

workers exposed to vinyl chloride, but if it is limited in terms of budget, time checks and spot
checks may not get in Indonesia, my opinion on a general check up periodically for
examination excretion of VC metabolites by finding thiodiglycolic acid in urine and
examined levels of enzymes in liver function tests such as AST, ALT and bilirubin of liver
dysfunction.
References

1. TJ Cheng et al. Urinary thiodiglycolic acid levels for vinyl chloride monomer-exposed
polyvinyl chloride workers. J Occup Environ Med.2001. 43(11):934-8.
2. Jennifer Schindler et al. The effect of genetic polymorphisms in the vinyl chloride metabolic
pathway on mutagenic risk. J Hum Genet. 2007. 52:448–455

3. Jing Zhang Yu. Interactions of chemical carcinogens and genetic variation in

hepatocellular carcinoma. World J Hepatol. 2010 . 2(3): 94-102

4. Attarchi Mir Saeed et al. Evaluation of liver enzyme levels in workers exposed to vinyl
chloride vapors in a petrochemical complex. Journal of Occupational Medicine and
Toxicology 2007. 2:6

5. Fromen O, et al. Immunoquantitation of von Willebrand factor (factor VIII -

relatedantigen) in vinyl chlorideexposed workers.Cancer Lett. 1992 Jan 31;61(3):201-6.

6. Y.L. Qiu et al. Messenger RNA expression and genetic polymorphisms of cell cycle control
genes and chromosomal aberrations in Chinese vinyl chloride monomer-exposed workers.
J Occup Environ Med .2011 Dec;53(12):1442-6.

7. International programme on chemical safety. Vinyl chloride, environmental health criteria

215. Available from http://www.inchem.org/documents/ehc/ehc/ehc215.htm. Sites on
march 22th 2013.

8. Peraturan Menteri Tenaga Kerja dan Transmigrasi Nomor PER.13/MEN/X/2011 tentang

Nilai Ambang Batas Faktor Fisika Dan Faktor Kimia Di Tempat Kerja. 2011.