Suitability of MDA, 8-OHdG and wild-type p53 as genotoxic biomarkers in metal (Co, Ni and Cr) exposed dental technicians: a cross-sectional study

The alloys Cobalt-chromium (Co-Cr) and Nickel-Chromium (Ni-Cr) are used extensively in dental medicine for removable partial dentures, porcelain-fused-to-metal crowns, and metal frames and particularly because they are, as compared to gold, bio-compatible, have a remarkable strength, corrosive resistant, and relatively less costly [1].. However, along with the benefits, during the manufacturing process, metal alloy exposure, as well as inaccurate working conditions and protection of workers, exposure to metals is hazardous to worker health. As more and more evidence found on metal-based nanoparticles in carcinogenicity, more studies have emerged, aimed to evaluate those nanoparticles’ genotoxicity and carcinogenicity, especially copper, chromium, nickel, and cobalt. The studies included those on epigenetic factors, for example, increased oxidative stress, abnormal apoptosis, and pro-inflammatory effects that involve those nanoparticles [2]..

The study, conducted by Hariyani et al., proved the impact of metal exposure significantly, ie the concentration of metal in the blood of dental technicians is higher than the control through research entitled, the effect of controlling the work environment on the levels of Co, Ni and Cr in the blood of the dental technicians [3]. The high concentration of Co, Ni and Cr is strongly associated with the formation of free radicals. It is highly reactive properties that can cause further oxidation of molecule in the vicinity. DNA damage due to ROS and DNA bases modifications results in altered genomic information. The damage may include deletions, insertions, point mutations, or chromosomal translocations that may lead to the oncogene activation as well as tumor suppressor gene inactivation, which have potential for initiating carcinogenesis [4]..

Reaction between ROS and membrane lipids can easily occur at high levels. This results in changes of membrane permeability, which results in chain reaction where a component of the cell membrane, the unsaturated fatty acid, is oxidized within varied pathological conditions. This condition is called as lipid peroxidation. The attack of radicals on polyunsaturated fatty acid residues of phospholipids leads to the formation of lipid peroxides, which furthermore may induce reaction with redox metals, and finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). As chromium (Cr) and cobalt (Co) perform redox-cycling reactions, nickel (Ni), a second group of metals, have the primary route of toxicity through glutathione depletion and the binding to sulfhydryl groups of proteins [5].. Oxidative attack on lipids leads produces reactive aldehydes as well. Among several different aldehydes that can be formed as secondary products of lipid peroxidation, MDA is the most mutagenic product of lipid peroxidation [6].

ROS react with DNA molecules causes modifications or damage to DNA structure and genomic instability affects genetic information contained inside. 8-OHdG is particularly a ROS-induced DNA base modification resulting from the attack of Hydroxyl radicals (OH) to guanine which triggers damage to the DNA. If it is not repaired, the damage will also be involved in cancer promotion and mutagenicity. Among the various types of oxidative DNA damage, 8-hydroxydeoxy guanosine (8-OHdG) formation is the most suitable marker of oxidative stress everywhere [7]. When DNA damage occurs, p53 protein activate s the p21 gene and the GADD45 gene for DNA repair. When DNA damage was severe, p53 protein activates the gene to trigger apoptotic processes [8]. If the cells cannot make improvements, there will be a disruption of DNA repair and result in cell death (pyknosis, karyorrhexis, karyolysis) [9].

ROS reaction with proteins results in oxidative modifications that causes catalytically less active enzymes or proteins that have higher susceptibility to proteolytic degradation. Protein damage may take place with carbonylation, thiol oxidation, fragmentation, side-chain oxidation, unfolding and misfolding, which may finally lead to activity loss [10]. In this case ROS are detrimental and induce cell apoptosis or necrosis. However, when the production of ROS does not change cell viability irreversibly, ROS may serve as primary messenger that modulates some cascades of intracellular signaling that leads to the progress of the cancer [5]..

The objective of this study was to review the suitability of MDA, 8-OHdG and p53 as Genotoxic Biomarkers, of Co, Ni and Cr dental technicians exposed in the workplace in contributing to the high incidence of cancer from metal exposure in the workplace.

Dental technicians that participated in this study were mostly in population of 19–29 years old or as much as 42.5% of the samples, while those of the age group 41–51 years were only 25%. Majortiy of the respondents were male, while women included in this study were few. (Fig. 1). The Cr concentration in dental technicians was the highest compare with Co and Ni concentration in dental technicians. Mean value of MDA and 8-OHdG in dental technicians were higher than controls. Comparative test revealed a significant difference with p value 0.000 (p < 0.05) between the values of the metal, MDA and 8-OHdG levels in the blood of the technicians as compared to the value of the metal in control.

There is significant correlation between 8- OHdG and heavy metals. The same result was also recorded on MDA and heavy metals. The observation revealed that Ni showed very weak correlation with both 8-OHdG (r = 0.06) and MDA (r = 0.09). Meanwhile, Co and Cr showed weak correlation with both 8-OHdG (Co: r = 0.337; Cr: r = 0.355) and MDA (Co: r = 0.337; Cr: r = 0.306). For PPE variable, 8-OHdG showed negative correlation (r = − 0.04) which means the higher 8-OHDG concentration, the less PPE be used (Table 1).

The correlation between heavy metal level and MDA concentration is illustrated in Fig. 1. Age and Range Based on Gender. This study involved a total sample of 40 respondents (32 males and 8 females), and 30 respondents belonged to control group with 18 males and 12 females. The sample group consisted of 17 respondents aged 19–29 years old (13 males and 4 females), 13 respondents aged 30–40 years old (10 males and 3 females), 10 respondents aged 41–51 years old (9 males and 1 females). The control group were dominated with respondents aged 19–29 years old (13 respondents with 10 males and 3 females), followed by 30–40 years old (10 respondents with 5 males and 5 females), and 41–51 years old (7 respondents with 5 males and 2 females). The following data were considered a balance proportion for both group.

Figure 2a shows the pattern of the relationship between Nickel and MDA. The higher the level of nickel, the higher the level of MDA. Figure 2c shows the pattern of the relationship between Chromium levels and MDA levels. The higher the level of Chromium, the higher the level of MDA. Thus the levels of Nickel and Chromium are positively related to MDA levels. While Fig. 2b shows an inverse relationship between Cobalt levels and MDA levels, the higher the Cobalt level, the lower the MDA level.

Figure 3 shows the higher levels of metals (Nickel, Cobalt, Chromium), the higher the level of 8-OHdG. A positive relationship was established between Nickel levels and 8-OHdG levels, between Cobalt and 8-OHdG levels, as well as between Chromium and 8-OHdG levels.

Figure 4 contains the correlation pattern between heavy metals and p53. It is stated that a high level of Chromium may also increase p53 level in blood. Meanwhile, a negative correlation was recorded for Cobalt; the higher Cobalt level, the less p53 expressed. A positive correlation also found between PPE with both Chromium and Nickel, yet interestingly, PPE and Cobalt level is showing a negative correlation (Fig. 5). Figure 5 shows pattern of relationship between metal content and PPE. It shows a positive correlation between PPE and metal (Cr and Ni), and a negative correlation for PPE and Co.

Figure 6 shows that the correlation pattern between 8-OHdG and p53 is negative. If 8-OHdG level is high, then the level of p53 is high. Conversely, if 8-OHdG level is high, p53 levels are low.

Table 1 show Mean, 95% confidence interval, and p-Value of each variable according to result of comparison test between exposed and control group. There are significant differences of metal concentration (Ni, Co, Cr) between the dental technicians and the control group. There are significant differences of MDA concentration between the dental technicians and the control group. There are significant differences of 8OHdG concentration between the dental technicians and the control group. There are significant differences of wild type p53 concentration between the dental technicians and the control group.

Previous research has concluded that dental technicians at Dental Laboratory in Surabaya have high Co, Ni and Cr levels. The high levels of metals in the blood of the dental technicians are due to the lack of use of self-protective devices so that exposure to metals can be absorbed through inhalation and skin during the manufacture of dental prostheses such as crowns, bridges and framework of partially released dentures [3]. Some of metal compounds have carcinogenic characteristics to human and animals as well. Metal-mediated free-radicals formation induces a variety of modifications to DNA bases, enhances lipid peroxidation, as well as alters the calcium and sulfhydryl homeostasis.

Cr actually plays role in human body to help the metabolism of glucose and fat. Cr exposure produces remarkable effect on human immune system, and, therefore, normally it is advised that exposure of the workers to chromic acid in electroplating industries should be reduced up to a minimum level. Co plays a role in the catalysis of vitamin B12 and is essential to the vitamin’s biological activity [13]. Ni. has an important role in iron absorption process in the body. However, nickel (Ni) is also reported as exerting intoxication to industrial workers. The risk of respiratory cancer is high in some certain groups workers who are exposed ro nickel, even though not all forms of Ni exposure are involved in such excessive risks [14].. In physiological conditions, these three metals are required only in very small quantities. The recommended reference values ​​are 0.09–4.18 μg / L for Ni, 0.04–0.64 μg / L for Co. and 0.1–0.2 μg / L for Cr [15]. The toxicity of Nickel, Cobalt, and Chromium is associated with its oxidation form. Cr (III), Cr (VI), Ni (II) is the most common and stable form of oxidation of Cr and Ni and is categorized as group 1 carcinogen associated with the incidence of lung cancer. Co. can also be found in oxidation forms of Co (II) and Co (III) and grouped as 2B group carcinogens.

This oxidation form acts as a catalyst in Fenton-like and Haber-weiss reactions to enhance Reactive Oxygen Species (ROS) formation in the form of hydroxyl radicals (OH) [16, 17]. Increased levels of metals can increase the process of free radical formation resulting in an imbalance between intracellular ROS and antioxidants and gives rise to a state of oxidative stress [18]. Increased production of ROS may worsen the dysfunction of mitochondria, in part due to peroxidation (oxidative damage of the lipids) [19].. Lipid hydroperoxides was produced through the reaction between free radical and double bonds of polyunsaturated fatty acids (PUFAs). It is a chain reaction from which free radicals supply were continuously provided due to its involvement in polyunsaturated fatty acid oxidation in the membrane that leads to damaged oxidative cells [20]. Malondialdehyde (MDA), a major secondary oxidation product of peroxidized polyunsaturated fatty acids, was believed to induced cytotoxic and mutagenic effects and might be related to cell aging and has a role in the onset of chronic morbidities such as cancer, atherosclerosis, inflammation, etc. [6, 21]. In this study statistical data obtained from the determination of MDA on serum dental technicians showed the Mean and SD higher than control.

Increased levels of oxidative stress in serum Dental technicians in this case represent an increase in local free radical production [21]. This indicated the activity of free radicals in cells as one of the clues of oxidative stress. Comparative statistical analysis between Co, Ni, and Cr levels in the exposed group with the control group, MDA, in blood serum of dental technicians showed significant differences (p < 0.05). This phenomenon showed that dental technician is one of the high risk occupations due to hazardous metal exposure. This high risk was indicated by the MDA value, where in normal individuals is less than 1.03 nmol/ml, and 2 times of that MDA value was considered pathologic. There is an important role that free radicals have in various diseases [22]..

When carcinogenic compounds entered the body and were activated, this compound would have covalent interactions with DNA to form DNA-adduct (modified DNA). This interaction could also lead to the breaking of the DNA chain. Among whole photooxidative DNA products, the biomarker 8-hydroxy-deoxyguanosine (8-OHdG) is sensitive and stable in the evaluation of DNA damage degree [23].. In our study the comparative statistical analysis between the levels of Co, Ni, and Cr in the group exposed to the control group, 8-OHdG, and wild type p53 protein in blood serum from dental technicians all showed a significant difference (p < 0.05). Although 8-OHdG level was not all proven to be positively correlated, only with Cr, and Co, and were negatively correlated with wild type p53, the comparative tests have shown the effect of high metal (Co, Ni and Cr) on the interactions with DNA to form DNA adducts, or 8-OHdG ensured that there has been damage to the DNA [24].

Associated with the analysis carried out on wild type p53 proteins, because p53 is considered a protein that has a function in the induction of apoptosis or arresting the cell cycle as a response to DNA damage, so that it maintains genetic stability of the organism. p53 shows genes that have the most mutations, especially in malignancies. Through the development of molecular biology at this time, it was revealed that one of the factors causing malignancy was the failure of the tumor suppressor gene, p53.

Physiologically if DNA damage occurs due to oxidative stress, the wild type p53 protein activates p21 and the gene for Catching Growth and Protein Inducible Protein Damage (GADD45) to enable DNA repair. Wild type p53 protein levels will increase, and play a role in DNA repair. If DNA damage is severe, cells will undergo apoptosis [25], or a p53 gene mutation occurs, which causes mutant p53 proteins to increase. In accordance with the research, it appears in this study that wild type p53 protein levels were lower than wild type p53 protein levels in controls and p53 negatively correlated with 8 OHdG. Decrease in wild p53 protein levels is caused by mutations in p53. Mutant p53 protein has a negative dominant effect on wild type p53 proteins by suppressing the wild-type p53 protein, which would result in the inactivation of p53 protein function. So there is a decreasing in wild-type p53 protein levels, and increasing in mutant levels of p53. Mutation of the p53 gene is believed to be associated with the result [26]. This finding supports the existing theory that p53 mutants have a negative predominant effect on wild type p53 protein, so if there is a decrease in wild p53 levels there may be an increase in p53 mutant levels [27, 28].

The oxidative damage also might affect the cell cycle, lead to mutations, and produce carcinogenesis because of enzymatic, endogenous, and nutritional antioxidant systems failure [29, 30]. According to Matsui, 8-OHdG played an important role in the early phase of carcinogenesis [31]. Enzyme couldn’t be able to recognize DNA molecules and trigger the formation of uncontrol growth cell that lead into malignancy. Oxidative DNA damage was mediated by reactive oxygen species and played an vital role in the occurrence of some diseases, including cancer [6]. With early detection of the risk, DNA damage was be prevented further so it did not cause cancer [32].

MDA, 8-OHdG and wild-type p53 can be used as genotoxic biomarkers in the metal exposed group, since they can accurately reflect the degree of oxidative damage.