Health Effects of Environmental Pollutants on Workers in the Libyan Plastic Factories, Part B: Based on Molecular Levels Characteristics

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 that expresses 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exposure to plastic and its solvent vapors during plastic manifesting on the blood DNA contents, blood samples were withdrawn from volunteers working in a plastics factory in Kasr Alakhyar city, Libya. Six different samples were collected from Workers 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


Introduction
Hundreds of thousands of people worldwide live or work in close proximity to plastic mills.Integrated plastic products generate chemical pollution such as plastic and other polymers containing compounds that can induce genetic damage (Alayeb, 2019;Legzdins et al., 1995;and Williams et al., 1990).Previous investigations demonstrated elevated DNA mutation rates near plastic mills but could not determine the importance of airborne or aquatic routes of contaminant exposure, or eliminate possible confounding factors such as nutritional status and disease burden (Manikkam et al., 2012;Somers et al., 2002;and Yauk & Quinn, 1996).To address these issues experimentally, laboratory mice were exposed in situ to ambient air in a polluted industrial area near plastic mills.Heritable mutation frequency at tandem-repeat DNA loci in mice exposed 1 km downwind from two integrated plastic mills was 1.5-to 2.0fold elevated compared with those at a reference site 30 km away.This statistically significant elevation was due primarily to an increase in mutations inherited through the paternal germ line.The results indicate that human and wildlife populations in proximity to integrated plastic mills may be at risk of developing germ line mutations more frequently because of the inhalation of airborne chemical mutagens (Somers et al., 2002).
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 have a negative impact on 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 10 mL syringe from volunteers working in plastic factory in Kasr Alakhyar city, Libya.Six different samples were collected from Workers 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 plyprobline tubes coated with EDTA to avoid the agglutination of blood and prepared for molecular analysis using randomly amplified polymorphic DNA (RAPD) and inter simple sequences repeats (ISSR).Molecular analyses were performed to assess the genotoxicity effect of plastic vapor on the molecular level.

Assessment of genotoxicity on the molecular level
The aforementioned six samples of the volunteers' blood were collected and subjected for molecular analysis using randomly amplified polymorphic DNA (RAPD).Peripheral blood leukocytes are a main source of animal genomic DNA, but sample collection is difficult as blood must be withdrawn from the animal.Blood contains a range of compounds like proteins, lipids, white blood cells, red blood cells, platelets, and plasma, which can contaminate the DNA sample.The primary contaminant of animal DNA extracted from blood samples is heme, the non-protein component of hemoglobin.

Genomic DNA extraction
Total DNA was extracted from one mL of blood sample using a Bioflux Kit (from china) as described in the kit manual.
Amplification was carried out in Strategene Robocycler Gradient 96, which was programmed for 40 cycles as follows: Denaturation (one cycle) 94 o C for 4 min., (40 cycles) of the following sequence 94 o C for 1 min.and 30 sec., 36 o C for1 min.and 30 sec., 72 o C for 2 min.and 30 sec., then extension (one cycle) 72 o C for 7 min.

Sample preparation
PCR-product 20 μL Loading buffer (6x) 5 μL The run was performed for one hour at 100 Volt using Biometra gel electrophoresis submarine (20 cm×10 cm).Bands were detected on UV-Transilluminator and photographed by gel documentation system (Biometra Bio Doc Analyzer-2000).The polymerase chain reaction products were resolved on T.A.E.agarose gels and recorded as (1) for the present bands and (0) for the absent ones.

Results and Discussions
Molecular analysis using randomly amplified polymorphic DNA (RAPD) were performed.DNA of the six volunteer's blood under investigation were extracted and subjected for molecular analysis using randomly amplified polymorphic DNA (RAPD) with nine ten-mer random primers, six out of the used primers only which produced scorable banding patterns.This technique was used to investigate whoever the exposure to plastic and its solvents vapors affect DNA or not.The six primers which have scorable banding patterns produced total number of 76bands, with average of 12.67 bands per primer.Out of the 76 produced bands, 44 monomorphic bands and 32 polymorphic bands.Sequences of some of these polymorphic bands seems to be modified as response for the exposure to plastic or its solvents vapors during the manifesting processes.

Primer Operon A-12
The results of primer (OP A-12) are shown in Figure (1) and Table (2).This primer resulted in kindly polymorphic products with the six blood samples under investigation.The PCR products using this primer resulted in a total number of ten bands with molecular sizes ranged from 320 to 1125 bp.All of the ten produced bands using this primer were polymorphic bands among the six blood samples under investigation.The results also showed that band with molecular size of 500 bp were presented only in control (volunteer never exposed to plastic or its solvents vapors) and it was absent in the blood samples of the exposed volunteers for the different periods under investigation.On the other hand, two bands were developed with length of 825 and 390 bp were present only in the blood sample of withdrawn from volunteer exposed to plastic or its solvents vapors for 12 years.These three aforementioned bands reflected a kind of trending modifications in the genomic DNA of the volunteers exposed to plastic or its solvents vapors for different lengths if compared with the banding patterns of the blood sample which withdrawn from control volunteer.These changes in the banding patterns between the blood samples withdrawn from the exposed volunteers and the blood samples of the non-exposed volunteer could be due to modification in the nitrogen bases unless the primer could not recognized the complement sequences and that is avoid the DNA amplification using polymerase chain reaction (PCR).

Primer Operon C-02
The results of primer (OP C-02) are shown in Figure (2) and Table (3).This primer resulted in kindly polymorphic products with the six blood samples under investigation.The PCR products using this primer resulted in a total number of fifteen bands with molecular sizes ranged from 370 to 1675 bp.eight bands out of the fifteen produced bands using this primer were monomorphic bands with lengths of 890, 820, 715, 680, 600, 540, 480, and 370 bp among the six blood samples under investigation.
The results also showed that the bands with molecular sizes of 1675 and 1090 bp were presented only in control (volunteer never exposed to plastic or its solvents vapors) but they were absent in the blood samples of the exposed volunteers for the different periods under investigation.
On the other hand, four bands were developed with length of 1490, 1125, 1015 and 420 bp were absent only in the blood sample of withdrawn from volunteer who never exposed to plastic or its solvents vapors (control) while they present in the blood samples of all the volunteers exposed to plastic or its solvents vapors from one year to 12 years exposure.Moreover, one band with length of 980 bp was present only in the blood samples of control and one year exposure volunteers but it was absent in the blood samples of the volunteers exposed to plastic or its solvents vapors for a periods of 3, 6, 7 and 12 years.
These seven aforementioned bands reflected a kind of trending modifications in the genomic DNA of the volunteers exposed to plastic or its solvents vapors for different lengths if compared with the banding patterns of the blood sample which withdrawn from control volunteer.These changes in the banding patterns between the blood samples withdrawn from the exposed volunteers and the blood samples of the non-exposed volunteer could be due to modification in the nitrogen bases unless the primer could not recognized the complement sequences and that is avoid the DNA amplification using polymerase chain reaction (PCR).

Primer Operon C-04
The results of primer (OP C-04) are shown in Figure (3) and Table (4).This primer resulted in kindly polymorphic products with the six blood samples under investigation.The PCR products using this primer resulted in a total number of seventeen bands with molecular sizes ranged from 80 to 1340 bp.seven bands out of the seventeen produced bands using this primer were monomorphic bands with lengths of 1340, 1270, 1210, 930, 860, 780 and 615 bp among the six blood samples under investigation.
The results also showed that the bands with molecular sizes of 430 and 240 bp were presented only in control (volunteer never exposed to plastic or its solvents vapors) but they were absent in the blood samples of the exposed volunteers for the different periods under investigation.
On the other hand, two bands were developed with length of 510 and 390 bp were absent only in the blood sample of withdrawn from volunteer who exposed to plastic or its solvents vapors for 7 and 12 years while they were absent in the blood samples of all the volunteers exposed to plastic or its solvents vapors from one year to 6 years exposure and control.Moreover, one band with length of 280 bp was absent only in the blood samples of control while it was present in blood samples withdrawn from volunteers exposed to plastic or its solvent vapors for periods of 1, 3, 6, 7 and 12 years.These five aforementioned bands reflected a kind of trending modifications in the genomic DNA of the volunteers exposed to plastic or its solvents vapors for different lengths if compared with the banding patterns of the blood sample which withdrawn from control volunteer.These changes in the banding patterns between the blood samples withdrawn from the exposed volunteers and the blood samples of the non-exposed volunteer could be due to modification in the nitrogen bases unless the primer could not recognized the complement sequences and that is avoid the DNA amplification using polymerase chain reaction (PCR).

Primer Operon E-19
The results of primer (OP E-19) are shown in Figure (4) and Table (5).This primer resulted in kindly polymorphic products with the six blood samples under investigation.The PCR The results also showed that a band with molecular size of 950 bp was present only in control (volunteer never exposed to plastic or its solvents vapors) and one year exposure but it was absent in the blood samples of the exposed volunteers for the periods of 3, 6, 7 and 12 years.
On the other hand, a band with molecular size of 190 bp was absent only in control (volunteer never exposed to plastic or its solvents vapors) and one year exposure but it was developed in the blood samples of the exposed volunteers for the periods of 3, 6, 7 and 12 years.These two aforementioned bands reflected a kind of trending modifications in the genomic DNA of the volunteers exposed to plastic or its solvents vapors for different lengths if compared with the banding patterns of the blood sample which withdrawn from control volunteer.These changes in the banding patterns between the blood samples withdrawn from the exposed volunteers and the blood samples of the non-exposed volunteer could be due to modification in the nitrogen bases unless the primer could not recognized the complement sequences and that is avoid the DNA amplification using polymerase chain reaction (PCR).

Primer Operon Q-18
The results of primer (OP Q-18) are shown in Figure ( 6) and Table ( 7).This primer resulted in kindly polymorphic products with the six blood samples under investigation.The PCR products using this primer resulted in a total number of ten bands with molecular sizes ranged from 75 to 1630 bp.Nine bands out of the ten produced bands using this primer were monomorphic bands with lengths of 1630,615,570,500,430,350,225,160 and 75 bp among the six blood samples under investigation.The results also showed that a band with molecular size of 690 bp was present only in control (volunteer never exposed to plastic or its solvents vapors) but it was absent in the blood samples of the exposed volunteers for the periods of 1, 3, 6, 7 and 12 years.This aforementioned band reflected a kind of trending modifications in the genomic DNA of the volunteers exposed to plastic or its solvents vapors for different length of periods if compared with the banding patterns of the blood sample which withdrawn from control volunteer.These changes in the banding patterns between the blood samples withdrawn from the exposed volunteers and the blood samples of the non-exposed volunteer could be due to modification in the nitrogen bases unless the primer could not recognized the complement sequences and that is avoid the DNA amplification using polymerase chain reaction (PCR).
In general the six primers which produced several differences in the banding patterns between the genomic DNA of the volunteer who never exposed to plastic or its solvents vapor (control) and the genomic DNA of the volunteers exposed to plastic or its solvents vapor for different period lengths during plastic manifesting.These results were in agreement with the findings of Wu et al. (2010) who stated that maternal exposure to DEHP was shown to increase DNA methylation and expression levels of DNA methyltransferases in mouse testis.Fetal testis was a main target for DEHP as evidenced in testicular dysgenesis syndrome due to a reduction in insulin-like hormone 3 (INSL3) expressions and testosterone production Also confirmed by Kundakovic et al. (2011) who reported that molecular mechanisms that underlie the long-lasting effects of BPA and phthalates continue to be elucidated, and they likely involve disruption of epigenetic programming of gene expression during development.It will be important to determine whether epigenetic markers in more accessible tissues correlate with epigenetic markers in target tissues.So, their results strongly imply that exposures to endocrine-disrupting chemicals (EDCs) may have cumulative adverse effects on future generations and that these effects could be mediated through epigenetic mechanisms.

Figure 1 .
Figure 1.Banding patterns of the six blood samples using RAPD-PCR primer (OP A-12) represented different exposure periods for plastic and its solvents vapors during manifesting processes

Figure 3 .
Figure 3. Banding patterns of the six blood samples using RAPD-PCR primer (OP C-04) represented different exposure periods for plastic and its solvents vapors during manifesting processes

Figure 6 .
Figure 6.Banding patterns of the six blood samples using RAPD-PCR primer (OP Q-18) represented different exposure periods for plastic and its solvents vapors during manifesting processes