Asphalts are complex hydrocarbon mixtures with molecular weights ranging from 500 to 2000, high boiling temperature (above 500°C) and carbon numbers predominantly higher than C25. Asphalts produced from the atmospheric and vacuum distillation of crude oil are used primarily in paving applications [1]. Asphalt typically contains about 80% by weight of carbon; around 10% hydrogen; up to 6% sulfur; small amounts of oxygen and nitrogen; and trace amounts of metals such as iron, nickel, and vanadium [2].

Asphalt fumes produced during heating of asphalts to facilitate their use commonly encountered by workers in road paving operations. They are complex mixtures of aerosols and vapors that contain various organic compounds including polycyclic aromatic hydrocarbons [3].

Polycyclic aromatic hydrocarbons “PAHs” are vapors result from incomplete combustion or pyrolysis of coal tar, pitch, coke and asphalt. They precipitate as particles or condense onto soot [4]. These fumes may be inhaled or deposited on skin or clothing [1].

The adverse effects of PAHs on humans are diverse [5]. PAHs have been reported to cause many skin disorders [6]; they were reported to be phototoxic [7] and verrucae inducer [8]. Numerous studies have described an excess risk of cancer among asphalt-exposed workers [9-12]. PAHs have been shown to be mutagenic in rat lung [13], reproductive and developmental toxicant [14] as well as genotoxic in humans [15].

Apoptosis is a natural, genetically controlled series of steps that occurs when a cell is old, unhealthy, or severely damaged. The end result is cell destruction. Apoptosis in the skin kills off harmed cells that they do not turn cancerous. In some cases, genetic mutations or other factors derail apoptosis. If this occurs; the cells can become “immortal” and continue to proliferate, resulting in skin cancer [16].

The process of apoptosis is controlled by important genes as P53 gene (tumour suppressor gene) and proteins such as Bcl-2 family i.e. several pro-apoptotic (Bax, Bak, Bad) and anti-apoptotic (Bcl-2, Bcl-xl) proteins and Caspases [17].

PAHs compounds have been shown to induce apoptosis in Hepa1c1c7 hepatoma cells [18], in human B cells [19], in macrophages [20] and in rat lungs [21]. Though numerous studies have described an excess cancer risk among asphalt workers, a causal relationship has not been established. Accordingly, the aim of this work firstly was to search for the dermatotoxic and carcinogenic risk associated with chronic PAHs exposure in asphalt workers and secondly to evaluate this cancer causal relationship through studying the PAHs effect on apoptosis by examining the expression of P53, Bax and Bcl-2 apoptotic proteins in skin specimens from road paving workers using immunohistochemical techniques.

The study was conducted on 152 male subjects classified into two groups: 122 road paving workers who were exposed nearly daily to PAHs asphalt fumes “PAHs exposed workers” and 30 age-matched nonpaving outdoor construction workers with no history of occupational PAHs exposure “control group”. The age of the road paving workers ranged from 30-55 years with a mean of 42 years and the exposure period to PAHs fumes ranged from 9–25 years with a mean of 11.5 years.

Prior to sampling; written consent was obtained from each participant after discussing the purpose and the protocol of the study. All procedures were conducted in accordance with a standardized human subject’s protocol that was approved by the ethics committee of Faculty of Medicine, Minia University.

Over 10 month period, for all subjects, a record of complete past medical history, occupational history and drug usage was compiled. Smokers were excluded. Complete general, local examination and photographing for any observed skin lesions were undertaken. 4 mm punch biopsies were taken from SCC lesions “if found” and from normal uninvolved forearm skin if no lesion were detected (non lesional skin). Patients with evident skin lesions were then referred to be properly managed. Biopsy specimens underwent routine haematoxylin and eosin light microscopic histopathological examination then evaluated immunohistochemically.

Immunohistochemistry was performed on paraffin wax embedded skin tissue according to Petrusz and Ordronneau [22]. 4 μm tissue sections were immunostained for P53, Bax and Bcl-2 proteins using a standard avidin-biotin-peroxidase method. Sections were dewaxed in two changes of xylene, hydrated in graded alcohol. Endogenous peroxidase was blocked with 0.3% H₂O₂(v/v) in methanol for 30 minutes at room temperature. Samples were then incubated overnight with rabbit monoclonal antibody to human P53 antibody (code no.: RMPD 016, ready-to-use 1ry antibody, DBS, CA, USA) which reacts with both the wild and the mutant types of human P53 protein, Bax epitope specific rabbit antibody (E3381, Spring Bioscience, CA, USA) and mouse monoclonal antibody to human Bcl-2 oncoprotein (code no.: PDM 016, ready-to-use 1ry antibody, DBS, CA, USA) at 4°C. Sections were then immersed in a biotinylated secondary antibody diluted at 1:100 for 30 minutes and in avidin biotinylated peroxidase complex for 60 minutes (code no.: # Kp-50L, DBS, CA 94566, USA). Slides were then treated with 100 μL of freshly prepared DAB (3’ 3’ diaminobenzidine) solution for 10 min. Finally, slides were rinsed in running water, dehydrated in graded methanol, cleared in three changes of xylene and mounted in DPX then examined. Negative control sections were obtained by omitting the specific primary antibody from the staining process.

The level of expression was evaluated quantitatively according to scoring system made by Liang et al [23]. Application of this system gives a score for both the degree of positivity (the number of positively stained keratinocytes/100 examined cells) and the degree of intensity of staining (Table 1).

Table 1: Scoring system for quantifying the level of P53, Bax and Bcl-2 expression in epidermal keratinocytes (Liang et al. 1999).

Statistical analysis of data was performed by using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA) for windows. Results were expressed as mean values (M) and standard deviation (± SD). Unpaired two-tailed Students’ t-test was performed to compare results. P Values of less than <0.05 was considered statistically significant.

Many dermatologic toxic symptoms and signs were observed in 71 PAHs exposed workers out of 122 examined (58.19%) as erythema (8 cases), itching (19 cases), excoriations (13 cases), chronic dermatitis (16 cases), chemical keratosis (9 cases) and keratoacanthoma (KA) (6 cases).

Nine out of 122 (7.38%) workers examined were found to have skin lesions in the form of 3 skin ulcers, 2 hyperpigmented lesions and 4 fungating masses. They gave history of working in asphalt paving for more than 15 years before noting skin lesions. They were exposed to PAHs fumes on a nearly daily basis. Lesions were limited to lower lip (Figure 1), ear pinna (Figure 2), back of scalp, knee and the nostrils entrance. Clinical as well as histopathological examination revealed squamous cell carcinoma (SCC) (Figures 3 and 4).

Figure 1: SCC of lower lip.

Figure 2: SCC of ear.

Figure 3: Moderately differentiated SCC showing an increased number of atypical keratinocytes and fewer horn pearls (H&E, x 400).

Figure 4: Poorly differentiated SCC with numerous atypical keratinocytes Occasional horn pearl can be seen within atypical keratinocytes (H&E, x 400).

The level of P53 expression in the epidermal keratinocytes of the control group ranged from 0.7-1.1 with a mean of 0.84 ± 0.16. Immunoreactive keratinocytes exhibited nuclear staining and were limited to the basal cell layer of the epidermis indicating wild type (diffuse pattern) P53 expression (Figure 5). While the level of wild P53 expression in the epidermal keratinocyte of the PAHs exposed uninvolved skin ranged from 0.62-1.9 with a mean of 1.3 ± 0.46 (Figure 6). Mutant type P53 (compact pattern) was over-expressed in SCC cases with a level ranged from 16.64-63.48 with a mean of 49.60 ± 32.7 (Figure 7). A significant increase in P53 expression level in epidermal keratinocytes of PAHs exposed normal uninvolved skin “non lesional” (t=2.51, P=0.04) and in SCC cases (t=4.79, p=0.003) was observed when compared with the control group (Histogram 1).

Figure 5: positive nuclear P53 expression in basal layer of epidermal keratinocytes of control group (Immunoperoxidase; x 200).

Figure 6: Increased nuclear P53 expression in epidermal keratinocyt- es of uninvolved skin of PAHs exposed group. The expression extended from basal to geanular cell layer (Immunoperoxidase; x; 400).

Figure 7: Positive nuclear P53 expression in SCC (Immunoperoxidase; x 200).

Bax was overexpressed in all PAHs exposed workers “either within PAHs exposed normal uninvolved skin or SCC lesions”. The expression was cytoplasmic and its level in epidermal keratinocytes was 2.2-2.46 with a mean of 2.3 ± 0.099, 1.57-3.75 with a mean of 2.81 ± 0.59 and 2.3-3.91 with a mean of 2.9 ± 0.02 in the control group, PAHs exposed normal uninvolved skin and SCC cases respectively (Figures 8, 9 and 10). A significant increase in Bax expression in epidermal keratinocytes of the PAHs exposed workers and in SCC cases was observed when compared with the control group (t=2.73, P=0.03) and (t=2.98, P= 0.01) respectively (Histogram 1).

Figure 8: Cytoplasmic expression pattern of Bax in basal cell layer of the epidermal keratinocytes of control group (Immunoperoxidase x 400).

Figure 9: Bax expression in all layers of epidermal keratinocytes of PAHs exposed un-involved group (Immunoperoxidase; x 400).

Figure 10: Higher Bax expression within SCC cells (Immunoperoxidase; x 200).

Bcl-2 expression of control group was confined to basal keratinocytes. They exhibited cytoplasmic staining with perinuclear enhancement (Figure 11) with mean expression level of 2.69 ± 1.7. In PAHs exposed normal uninvolved skin; Bcl- 2 was down regulated with a mean level of 1.3 ± 1.2 (Figure 12). On other hand; none of the 9 tested SCC cases were positive for Bcl-2 (Figure 13). A significant decrease in Bcl-2 expression in epidermal keratinocytes of PAHs exposed subjects was noted (t=2.49, P=0.047) (Histogram 1).

Figure 11: Cytoplasmic expression of Bcl-2 of basal cell layer in control skin (Immunoperoxidase; x 400).

Figure 12: Dowenregulated Bcl-2 expression in PAHs exposed workers (Immunoperoxidase; x 400).

Figure 13: Negative Bcl-2 expression in SCC (Immunoperoxidase; x 400).

Number of positively immunostained cases, immunostaining intensity and the expression score for P53, Bax and Bcl-2 in both PAHs exposed normal uninvolved skin and SCC cases are illustrated in Tables 2 and 3 and Figures 14A and 14B.

Table 2: Immunostaining intensity of P53, Bax and Bcl-2 in uninvolved skin and SCC cases of PAHs exposed group.

Table 3: Apoptotic markers expression score in uninvolved skin and SCC cases of PAHs exposed group.

Figure 14: Immunostaining intensity of P53, Bcl-2 and Bax in uninvolved skin (A) and SCC (B) cases of PAHs exposed group.

Occupational exposure to hot asphalt may increase the risk of cancer (lung, stomach, bladder, leukemia and non-melanoma skin cancer) among asphalt workers [24]. Changed expression of apoptosis related molecules could be detected during the process of malignant transformation and in malignant cells [25]. As dermatotoxic and carcinogenic potentials of PAHs are not fully clarified and mechanisms involved in PAHs-induced apoptosis are less characterized; so this work aimed to study chronic toxic effects of PAHs fumes on skin and to search for different cellular pathways of apoptosis underlying malignant transformation through studying P53, Bax and Bcl-2 apoptotic protein expression in skin of a population of road paving workers to assess the toxic role of PAHs on apoptosis using immunohistochemical techniques.

The observed increased toxic skin symptoms in PAHs exposed workers agree with what reported by [6-8]. This may be explained on the basis that asphalt alters cutaneous barrier integrity and induce skin disease. Results also proved an association between cancer risk and exposure to PAHs fumes where 9 chemical keratosis cases (with precancerous potential) and 9 SCC cases were diagnosed which in line with [26,27].

Apoptosis which is equated to programmed cell death is an evolutionary conserved cell death process that plays a major role during normal development, homeostasis and elimination of potentially dangerous cells. The ordered execution of this internal death program leads to typical morphological and biochemical changes [28]. Dysregulation of the apoptotic process may result in a wide range of pathological conditions as cancers, autoimmune diseases, and viral infections [29].

In this study; PAHs were found to induce activation of apoptosis where both wild type P53 (tumour suppressor gene) and Bax protein (pro-apoptotic protein) were over-expressed and Bcl-2 (the major antiapoptotic protein) was downregulated which agree with [30-32]. The activated apoptosis act as a defense mechanism to prevent PAHs toxic effect as it helps exposed workers to get rid of harmed cells and to prevent progression to carcinoma. Increased P53 expression level suggests that PAHs induced apoptosis is triggered by the induced DNA damage in P53-dependent pathway. P53 in its natural form (wild-type) upregulates P21 causing cell cycle arrest to allow repair of damaged DNA [33]. Road paving asphalt fume was found by [34] to be mutagenic and induced DNA damage and adduct formation. DNA damage initiates a number of cell signaling pathways that may result in cell cycle delays, senescence or cell death. The key point of these changes is the tumour suppressor P53 [35]. Following DNA damage P53 is phosphorylated thereafter translocated to the nucleus where it may result in cell cycle arrest, allowing time for DNA repair or triggering apoptosis [36]. If damage is extensive, P53 induces apoptotic cell death by downregulating Bcl- 2 and upregulating Bax expression [33]. Comparatively, we were able to further substantiate this hypothesis as we found an increased level of Bax which is involved in triggering the apoptotic process as Bax is known to be a downstream target of P53 and is often induced and/or translocated to mitochondria as a part of the apoptotic process [37]. The enhanced apoptosis was also confirmed from the observed lowered expression level of Bcl-2. Bcl-2 is a proto-oncogene that encodes the largest known anti- apoptotic protein. Bcl-2 regulates apoptosis through the formation of homodimer proteins with the other proteins belonging to the Bcl-2 group, especially the pro-apoptotic protein Bax [38].

PAHs-induced apoptosis may also have a role in cancer initiation and promotion. Results showed significantly increased expression levels of mutant P53 in SCC cases. The observed higher level of mutant P53 accompanied with high Bax expression may explain the aggressive behavior of SCC as the mutant type P53 protein was unable either to induce apoptosis or to suppress SCC development and progression [39]. Mutation in P53 gene leads to the loss of its repair function and thus apoptosis resistance of the DNA-damaged cell and hence cancer development [40]. Hussain and Harris [41] found that mutations in P53 gene are the most frequently observed genetic alteration in cancer. P53 mutation may occur due to chromosomal instability, reactive nitrogen and oxygen species and formation of reactive metabolites which induce G to T transversion mutations [42]. The lack of Bcl-2 expression in SCC confirms the hypothesis that most of tumor cells originate from the suprabasal keratinocytes which normally have negative Bcl-2 expression [43]. The negative Bcl-2 expression in SCC was unexpected as Bcl-2 is known to protect tumor cells from apoptosis [44]. This may be possibly compensated by over expression of other anti-apoptotic proteins (not included in the study). So, it is suggested that disturbed apoptosis and SCC formation in PAHs exposed workers is not Bcl- 2 dependent. Although the relatively high expression level of Bax; it appears to be unable to suppress the tumor progression. This may be due to expression of functionally inactive Bax [45]. Another explanation to tumor formation was reported by Holme and Solhaug [46] where they stated that although apoptosis of cells exposed to PAHs is considered to function anti-carcinogenic since cells with extensive DNA damage will be removed, it is possible on other hand that removal of these cells may give survival and proliferating signals to surrounding cells with less DNA damage, increasing their probability of having mutations and give promotion signals to cell proliferation and hence cancer formation.

It could be concluded that chronic exposure to asphalt fumes may increase the risk for developing dermatotoxic and/or cancer which could be explained by PAHs exposure can disturb P53, Bax and Bcl- 2 apoptotic protein controlling apoptosis. The asphalt fumes could be considered as a risk factor disturbs apoptosis; however this disturbance needs further thorough investigation on other apoptotic proteins as Bcl-xl, Caspases and Fas.

Special thanks to Dr. Ghada Ali Nassef, lecturer of Dermatology, STDs and Andrology, Faculty of medicine, Minia University for her role in performing the clinical dermatological examination and management of diagnosed cases.