Health Effects of Environmental Pollutants on Workers in The Libyan Plastic Factories, Part A: Based on Biochemical Analysis

This study aimed to disclose the reasons for the health damage and heritable genetic mutations among workers in plastic manufactories due to negative environmental impacts. Moreover, this study may constitute an initial database expressing the negative impact on the environment in the plastics factories that may have a negative impact on workers. To study the effects of long-term exposure to plastic and its solvent vapors during plastic manifesting on the blood protein contents, blood samples were withdrawn from volunteers working in a plastics factory in Kasr Alakhyar City, North-west Libya. Six different samples were collected from Clarkes exposed to plastic vapor for different periods as follows, control (did not expose to plastic vapor), one-year exposure, three years exposure, six years exposure, seven years exposure, and twelve years exposure. Blood samples were collected and subjected to protein electrophoresis and isoenzymes analysis for esterase and peroxidase. The results of protein electrophoresis indicated that a total number of 14 protein bands were resolved on polyacrylamide gel electrophoresis ranging from 16.5 KDa to 105 KDa. Five bands out of the resulted from 14 bands were considered common bands with molecular weights of 105, 98, 85, 63, and 16.5 KDa. (i


Introduction
Live and working near plastic mills, hundreds of thousands of people worldwide.Integrated plastic goods manufacture chemical waste such as plastics and other polymers which contain compounds that can cause genetic damage (Williams et al., 1990).Chemical mutagens that contaminate atmospheric and ecosystems cause a human and wildlife genetic risk.Nesting near steel mills on the Great Lakes, Herring gulls (Larus argentatus) showed higher germline mutation rates at minisatellite DNA loci than those at rural sites and increased mutation frequency with colony proximity to integrated plastic mills.It was believed that inhaled airborne contaminants released from plastic factories, such as polycyclic aromatic compounds, are largely responsible for mutation induction; nevertheless, as contributing factors, contaminants in the aquatic food web and variations in disease and nutritional quality among gull colonies could not be excluded.Therefore, it was not possible to determine the role of air pollution in the development of germline mutations and the threat to people living near plastic mills.
Use in situ exposed sentinel laboratory animals is an effective research approach to determining air pollution hazards as it blends monitored elements of laboratory studies with direct exposure to rates of environmental pollution.Compare germ line ESTR mutation rates in laboratory mice exposed to ambient air at an industrial site near integrated plastic mills with those exposed at a rural reference site, with the goal of evaluating industrial air pollution inhalation as a route of chemical mutagen exposure (Larsen and Nielsen, 2012).
This study aimed to disclose the reasons for the health damage and heritable genetic mutations among workers in plastic manufactories due to negative environmental impacts.Moreover, this study may constitute an initial database express the negative impact to the environment in the plastics factories that may harm workers.

Materials and Methods
To study the effects of long term exposure to plastic and its solvents vapors during plastic manifesting on the blood protein contents, blood samples were withdrawn using 10ml syringe from volunteers working in a plastics factory in Kasr Alakhyar city, North-west Libya.Six different samples were collected from Clarkes exposed to plastic vapor for different periods as follow, control (did not exposed to plastic vapor), one-year exposure, three years exposure, six years exposure, seven years exposure and twelve years exposure.
Blood samples were collected in polypropylene tubes coated with EDTA to avoid the agglutination of blood and prepared for protein electrophoresis and isoenzymes analysis.

Assessment of Genotoxicity on The Protein Level
The aforementioned samples of the volunteers' blood were collected and subjected to biochemical analysis using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein electrophoresis and isozymes electrophoresis.The SDS-PAGE included extraction of soluble protein.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) used according to Laemmli (1970) method of for the aforementioned six blood samples.

Water-Soluble Protein Extraction
Extraction of water-soluble proteins was performed as the following: Five ml of each blood sample was mixed in pre-chilled mortars with 10 ml of water-soluble extraction buffer and pestles.
Samples were transferred to Eppendorf tubes and incubated at 4 o C in the refrigerator overnight, then centrifuged for 20 minutes at 12,000 rpm at 4 o C, supernatant containing water-soluble proteins fraction were transferred to clean tubes and stored at -20 o C .

Polyacrylamide Gel Electrophoresis
Polyacrylamide gel electrophoresis was performed as follow: Gels were poured simultaneously to a height of 1.5 cm below the bottom of the comb.Gels were overlaid with isopropanol and left to polymerize.Then isopropanol was removed before the stacking gel was poured.

b) Stacking gel
Acrylamide stock (for stacking gel) 2.66 ml This stacking gel solution was quickly poured off over the two resolving gels, and then combs were used.Gels were left to polymerize.

Sample Preparation
A volume of 50 µl extracted protein for each sample was added to equal volume of loading buffer (2x for the water-soluble fraction and 1x for the water-no soluble fraction), 10 µl ß-Mercaptoethanol was added to each sample.Samples were boiled for 10 min in a water bath, then 10 µl bromophenol blue was added for each sample.25 µg protein of each sample was loaded .

Gel running and Staining
Four liters of pre-cold run buffer were poured into the running tank; cooler circulator was used during the run to keep the gels cold.Gels were run at 100 vol.for 15 min, and then the voltage was raised up to 250 vol.till the samples reached one inch from the bottom of the gel.Gels were removed from the apparatus and placed in tries for staining.Gels were covered in the tries with staining solution.Gels were gently agitated overnight.

Staining solution consisted of;
Commassie brilliant blue-R250 1.0 g Methanol 455 ml Acetic acid 90 ml H2O (dd) 455 ml After removing the staining solution, gels were covered with a distaining solution which consisted of ‫ك‬ Banding patterns of each sample were recorded as (+) for the present band and (-) for the absent ones.Also, the bands' intensities were recorded and classified as (+) for faint bands, (++) for moderate-intensity and (+++) for heavy or dark bands.

Isozymes Electrophoresis
The volunteers' blood samples from the six exposure periods were collected and subjected for biochemical analysis using polyacrylamide gel electrophoresis (PAGE) protein electrophoresis for isozymes electrophoresis.The PAGE included-water soluble protein extraction.Homogeneous (native) polyacrylamide gel electrophoresis (PAGE) was performed according to Stegmann et al. (1983).

Peroxidase Isozymes
Peroxidase isozymes were performed and its banding patterns were developed from watersoluble protein extracts of the six samples according to Larsen and Benson (1970), using the following developer solution: Benzidine-dihydrochloride 16 mg Glacial acetic acid 1 ml Hydrogen peroxide (H2O2) 0.5 ml H2O 100 ml The reaction was stopped by using 200 ml of tap water.
Banding patterns of each sample were recorded as (+) for the present band and (-) for the absent ones.Also, the bands' intensities were recorded and classified as (+) for faint bands, (++) for moderate-intensity and (+++) for heavy or dark bands.

Esterase Isozymes
Esterase isozymes were performed and its banding patterns were developed using water soluble protein extracts of the six aforementioned blood samples according to Scandalios (1984)  The reaction was stopped by using 200 ml of tap water.
Banding patterns of each sample were recorded as (+) for the present band and (-) for the absent ones.Also, the bands' intensities were recorded and classified as (+) for faint bands, (++) for moderate-intensity and (+++) for heavy or dark bands.

Results and Discussion
Results of the Protein electrophoresis and isoenzymes analysis are presented the assessment of genotoxicity on the protein level.On this level sodium dedocayl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two isozymes, peroxidase and esterase were analyzed.Their banding patterns were recorded and used to assess the genotoxicity of plastic and solvents vapors on the base of the variations in the banding patterns among the six blood samples in the aforementioned techniques.

Sodium Dedocayl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Total water-soluble proteins were analyzed electrophoretically using sodium dedocayl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).The protein banding patterns of the six blood samples under investigation were developed and recorded as described previously.The results are shown in Figure ( 1) and presented in Table (1).The results indicated that a total number of 14 protein bands were resolved on poly acrylamide gel electrophoresis ranged from 16.5 KDa to 105 KDa.Five bands out of the resulted 14 bands were considered as common bands with molecular weights of 105, 98, 85, 63 and 16.5 KDa.(i.e.these bands were presented in all the six blood samples under investigation).
The results also showed that the band with molecular weight 79 KDa was absent in control (a volunteer never exposed to plastic or its solvents vapor) but it present in all blood samples from volunteers exposed for different periods to plastic or it solvents vapors, suggesting that this newly synthesized protein was produced in the blood as response for the exposure to plastic or its solvents vapor during plastic manifesting.
Another three protein bands with molecular weights of 74, 49, and 23 KDa were absent in control (a volunteer never exposed to plastic or its solvent5s vapor) and all samples which extracting from volunteers spent a period of time for 7 years or less exposed for plastic or its solvents vapors but it present only in blood samples from volunteer exposed for 12 years (the longest investigated exposure period) to plastic or it solvents vapors, suggesting that these bands could be induced after this long period exposure.

Alayeb, 2019
Faculty of Marine Resources, Alasmarya Islamic University, Libya.One more bandwidth molecular weight of 52 KDa was absent in control (a volunteer never exposed to plastic or its solvent5s vapor) and all samples which extracting from volunteers spent a period of time for 6 years or less exposed for plastic or its solvents vapors but it present only in blood samples from volunteers exposed for 7 and 12 years (the two longest investigated exposure periods) to plastic or it solvents vapors, suggesting that these bands could be induced after this long period exposure.

Health Effects of Environmental Pollutants on Workers …………………………
Faculty of Marine Resources, Alasmarya Islamic University, Libya.Moreover, there were two bands with molecular weights of 72 and 32 KDa which presented only in control only but they were absent in all of the blood samples from the volunteers who exposed to plastic or its solvents vapors.It possible to conclude that the absence of these two bands referred to the exposure to the vapors .

E-23
Finally, there were two bands with molecular weights of 57, and 42 KDa which presented in control and all the exposed volunteers except the 12 years exposure where it was absent in the 12 years exposure.It possible to the absence of these two bands referred to the exposure for a very long time to the vapors.

Isozymes Electrophoresis
Isozymes (also referred to as isoenzymes) are enzymes that differ in the sequence of amino acids but catalyze the same chemical reaction.Such enzymes usually show different kinetic parameters (e.g.different Km values) or different regulatory characteristics.The presence of isozymes enables metabolism to be fine-tuned to meet the specific needs of a given tissue or stage of development Isozymes (or isoenzymes) in biochemistry are enzyme isoforms (closely related variants).In many cases, homologous genes that have diverged over time are coded for them.While allozymes are enzymes from different alleles of the same gene and isozymes are enzymes from different genes which process or catalyze the same reaction, the two terms are generally used interchangeably.
Isozymes usually result from gene duplication, but can also result from polyploidization or hybridization of nucleic acid.If the new variant's function remains identical to the original over an evolutionary period, then it is possible that one or the other will be lost as mutations occur, resulting in a pseudo-gene.However, if the mutations do not necessarily stop the enzyme from functioning but instead alter either its function or gene expression pattern, then both variants can be preferred by natural selection and specialize in different functions.They can be articulated, for example, at various developmental stages or in different tissues.
Allozymes can be the result of point mutations or insertion-deletion (indel) events affecting the gene's DNA coding sequence.There are three things that can happen to a new allozyme, like any other new mutation: -The new allele is most likely to be non-functional in which case it is likely to result in low fitness and will be eliminated by natural selection from the population.-Instead, if, for instance, the modified amino acid residue is in a relatively unimportant part of the enzyme far from the active site, the mutation may be strictly neutral and susceptible to genetic drift.-In rare cases, the mutation can lead to an enzyme that is more effective and capable of catalyzing a slightly different chemical reaction, in which case the mutation may lead to an increase in fitness and benefit natural selection.

Peroxidase Isozymes
Peroxidases are a broad enzyme family usually catalyzing a type reaction: The optimal substrate for many of these enzymes is hydrogen peroxide, but others with organic hydroperoxides such as lipid peroxides are more active.Peroxidases in their active sites may contain a heme cofactor, or residues of redox-active cysteine or selenocysteine.
The nature of the electron donor depends heavily on the enzyme's structure.For example, as donors of electrons and acceptors, horseradish peroxidase can use a variety of organic compounds.Horseradish peroxidase has an active site that is accessible and many compounds can enter the reaction site.
The that donate electrons are very unique to an enzyme such as cytochrome c peroxidase because there is a tightly closed active site.Although the exact mechanisms have yet to be established, peroxidases are known to play a role in raising the defenses of a plant against pathogens (Zhang et al., 2015).Peroxidases are sometimes used as a marker of histology.Cytochrome c peroxidase is used in cytochrome oxidase as a soluble, easily purified template.The family of glutathione peroxidase is made up of eight known human isoforms.Glutathione peroxidases are involved with both hydrogen peroxide and organic hydroperoxide substrates as an electron donor.It has been shown that Gpx1, Gpx2, Gpx3 and Gpx4 are selenium-containing enzymes, while Gpx6 is a selenoprotein in humans with rodent homologs derived cysteine.Amyloid beta was shown to have peroxidase activity when bound to heme.Haloperoxidases are a common class of peroxidoses.This group can form reactive species of halogen and, as a result, natural substances of organohalogen.Many protein sequences for peroxidase can be found in the Peroxidase database.
Obtained results are shown in Figure (2) and presented in Table (2), the zymogram with eight different peroxidase isozymes were detected using the substrate and developer solution specific for peroxidase, these eight bands suggested that there are eight alleles conferring peroxidase isozymes in human.The eight peroxidase isozymes were exhibited with different intensities among the six blood samples under investigation.On the other hand, relative mobility was presented between these peroxidase isozymes.The relative mobility reflecting different sizes of peroxidase molecules.Peroxidase 1 and peroxidase 3 were absent only in the control sample but they presented with different intensities in all the exposed volunteers.These results suggested that peroxidase 1 and peroxidase 3 were positively regulated as response for the exposure to plastic or its solvent vapors.
On the contrary, peroxidase 2 and peroxidase 4 were presented with a very dark intensity only in the control blood sample but they were absent in all the blood samples of the exposed volunteers.These results suggested that peroxidase 2 and peroxidase 4 were negatively regulated as response for the exposure to plastic or its solvent vapors.Furthermore, peroxidase isozyme 6 was present in blood samples collected from volunteers exposed for 3 years or more to plastic or its solvents vapors, but it was absent in both blood samples of control and the blood sample of the volunteer exposed for one year to the vapor.These results suggested that peroxidase 6 was positively regulated as a response for 3 years or more exposure to plastic or its solvent vapors.
Finally, peroxidase isozymes 5 and 8 were present in all the blood samples reflecting that there was no effect for the exposure to plastic or its solvent vapors on the regulation of these two peroxidase isozymes .

Esterases Isozymes
An esterase is an enzyme of hydrolysis that separates esters into an acid and an alcohol in a chemical reaction called hydrolysis with water.There are a wide range of different esterases that vary in their specificity of substrates, their structure of proteins, and their biological function.
The results are shown in Figure (3), and Table (3), the represented zymogram with five different esterase isozymes were detected using the substrate and developer solution specific for esterase, these five bands suggested that there are five alleles or loci conferring esterase isozymes in these six blood samples which were withdrawn from the volunteers under investigation.The five esterase isozymes were exhibited with different intensities between the six volunteers under investigation.On the other hand, relative mobility was presented between these esterase isozymes.The relative mobility reflecting different sizes of esterase molecules.Esterases 1 and 2 were presented in all the six blood samples under investigation with similar intensities (the darkest intensity), for that, they considered as common bands indicating no effects of plastic or its solvents vapors during manifesting on these two esterase isozymes.The results indicated also, that esterase isozyme 3 was presented only in the control, one-year exposure and 3 years exposure blood samples.On the other hand, it was absent in the blood samples of 6, 7, and 12 years of exposure volunteers.This banding pattern indicated that a negative effect for the exposure to plastic or its solvents vapors for a period of time longer than 3 years was occurred in esterase isozyme 3. Esterase 4 was presented in all the six blood samples of the volunteers under investigation with different intensities.
Esterase isozyme 5 was presented in all samples except the sample which withdrawn from the volunteer exposed to the vapors for a period of time of 12 years.This result indicated that the negative effect resulted from the exposure to plastic or its solvents vapors during plastic manifesting occurred after a long time of exposure, longer than 7 years exposure.These results could be explained by the findings of (Dolinoy et al., 2007), which stated that epigenetic effect bioaccumulation after exposure to BPA (plastic) could be reversed/ eliminated.Differential DNA methylation after exposure of gestating female rats to a mixture of BPA and phthalate was also confirmed to be transgenerational, but the synergistic effect of both BPA and phthalate remains to be determined.
The increasing evidence suggests that epigenetics has the potential to develop biological markers to predict that chemicals would put exposed subjects at risk and which individuals would be more susceptible to disease.t is still important to note that to better understand the nature of epigenetic changes and the health effects of toxic influences on these diseaseassociated epigenetic modifications, the mechanisms by which environmental toxicants modulate the epigenetic landscape of individual cells have yet to be elucidated.Better-defined mechanisms can lead to better detection of the toxic potential of environmental chemicals such as BPA and phthalates and allow more tailored and effective strategies for disease prevention.
For human studies, it will be important to use laboratory methods with increased accuracy, tolerance and coverage to detect epigenetic changes as early as possible and well in advance of disease diagnosis.Now new technologies available to allow the global study of epigenetic changes, which can provide insight into the degree and patterns of changes between normal human and diseased tissues.It is necessary to consider suitable in vitro models.In this sense, human embryonic stem cells can be extremely useful in enhancing understanding of epigenetic effects on human growth, health and disease, as the creation of in vitro embryonic bodies is very similar to the early stage of embryogenesis (Li et al., 2006;and Chen et al., 2011).Also, Manikkam et al. (2012) confirmed that the transient exposure of female rats to a plastic mixture (BPA and phthalates) during the embryonic sex determination cycle was demonstrated to encourage transgenerational female early-onset puberty (F3 generation) and decrease the pool size of primordial ovarian follicles.Spermatogenic cell apoptosis was also transgenerationally affected, and all exposed lineage males found differential DNA methylation of the F3 generation sperm promoter regions.

Conclusion
This study aimed to clarify the dangerous effects of different periods of exposure to plastic and its solvents vapors during plastic manifesting in the form of the changes in the blood protein contents and the modification in the genomic materials.To reach this target, blood samples were withdrawn from volunteers working in a plastics factory.Six different samples were collected from Clarkes exposed to plastic vapor and their solvents vapors for different periods as follow, control (did not exposed to plastic vapor), six months exposure, one-year exposure, three years exposure, six years exposure and twelve years exposure.The results of protein electrophoresis indicated that a total number of 14 protein bands were resolved on polyacrylamide gel electrophoresis ranged from 16.5 KDa to 105 KDa.Five bands out of the resulted 14 bands were considered as common bands with molecular weights of 105, 98, 85, 63 and 16.5 KDa (i.e.these bands were presented in all the six blood samples under investigation).The results also showed that too many changes were recorded on the protein banding patterns between the control volunteer and those who exposed to plastic and its solvents vapor for different exposure periods.These changes in proteins suggested that a modification in gene excretion was occurred as a response for the different exposure time to plastic during manifesting.Also, in esterase and peroxidase isozymes changes in the banding patterns between the control volunteer and those who exposed to plastic and its solvents vapor for different exposure periods.These changes in proteins suggested that a modification in gene excretion was occurred in the genes conferring these isozymes as response for the different exposure periods to plastic during manifesting.
agitated for 1hr in the distaining solution.The distaining solution was changed several times until it becomes clear when removed from the tray and the gels background becomes clear and bands were clear enough for a photograph .

Figure 1 .
Figure 1.The banding patterns on polyacrylamide gel electrophoresis for the extracted water-soluble proteins from six volunteers blood of exposed to plastic and its solvents for different periods.

Figure 2 .
Figure 2. The Zymogram of peroxidases as detected using electrophoresis analysis for extraction from six blood samples of control and volunteers exposed for different times to plastic or its solvents vapors during plastic manifesting

Figure 3 .
Figure 3.The Zymogram of Esterases as detected using electrophoresis analysis for extraction from six blood samples of control and volunteers exposed for different times to plastic or its solvents vapors during plastic manifesting

Table 1 .
The banding patterns on polyacrylamide gel electrophoresis for the extracted water-soluble proteins from six volunteers blood exposed to plastic and its solvents for different periods * Molecular weight(KDa)

Table 2 .
The Zymogram of peroxidases as detected using electrophoresis analysis for extraction from six blood samples of control and volunteers exposed for different times to plastic or its solvents vapors during plastic manifesting * .

Table 3 .
The Zymogram of Esterases as detected using electrophoresis analysis for extraction from six blood samples of control and volunteers exposed for different times to plastic or its solvents vapors during plastic manifesting * Represented signs as; (+) for present and (-) for absent bands and the number of + representing the bands' intensities.