Photosensitization Studies in Selected Dyestuffs

There are various dyestuffs species to produce reactive oxygen such as singlet oxygen O2 and super oxide radicals. Irradiation with sun light in vitro, the dyestuff like benzanthrone, metanil yellow and paminodiphenylamine were found to produce reactive oxygen species such as singlet oxygen (1O2) and/or superoxide radicals (O2 ). However benzanthrone produces detectable amount of 1O2 Although metanil yellow and p-aminodiphenylamine (p-ADPA) did not produce detectable amounts of 1O2 under similar conditions. The above dyestuffs are routinely used in textiles, cosmetics, detergents, leather industries as well as food additives. In view of the fact that workers exposed to some of these chemicals during manufacture, pulverization and storage develop edema and even skin cancers in the exposed areas of skin. We believe that activated oxygen species may play a major role in the development of these symptoms.


Introduction
Production of synthetic dyes in India is about 25000 metric tons every year 1 . Which are Industrial health hazards to people 2 . Dyestuff industries have occupied a prominent place in the industrial organization of the modern world. One of the major routes to incorporate dyestuffs in our biological system is by absorption through skin. Some dyes have different degrees of binding ability with protein, collagen and lipids [3][4] . Study shows that toxic effect of these synthetic dyes are observed in the presence of the ultraviolet component of sunlight (285-400 nm) 5 . Photosensitization reactions in our analysis is oxygen dependent in which a photo excited dye transfers energy to molecular oxygen to produce activated oxygen species like singlet oxygen ( 1 O 2 ), super oxide radical (HO 2 or (O 2 -), hydroxyl radical (OH) and hydrogen peroxide. The activated oxygen species contribute to oxidative degradation of protein, enzymes, lipids and other target molecules of cellular systems [6][7][8] and are also responsible to act as tumor promoters in the development of cancer 9 .
In present study the reason for taking benzanthrone (7H-benz(de)anthrance-7-one) is used in the synthesis of many vat and disperse dyes. It has been observed that workers coming into contact with it during manufacture, pulverization and storage develop itching, burning sensation, erythema, roughness, dryness and pigmentation of the skin [10][11] . Orange II [monoazo, C.I. acid yellow, orange 7 (15510)], a sodium or calcium salt of p-(2-hydroxyl-1-naphthalenyl) azo benzenesulfonic acid is used in dyeing of paper, soaps etc 12 . A blend of orange II with metanil yellow under the trade name to cause deleterious effects in our biological system 13 . Metanil yellow [monoazo, C.I. acid yellow 36 (13065)], a sodium or calcium salt of m-(p-anilinophenyl-azo) benzenesulfonic acid, is used in the coloring of foodstuffs, soaps, shoe polishes, wood stains, paper staining and cosmetics. p-Aminodiphenylamine (p-ADPA) and metanilic acid are important metabolites of metanil yellow 14 and are considered to be an active ingredient of metanil yellow induced toxicity 15 . p-ADPA is used as an intermediate for the synthesis of photographic chemicals, and the manufacture of dyeing reagents [16][17] . It is also used as an ingredient in oxidizing types of hair dyes 18 . Its properties of skin irritation and sensitization are well known 19 .
Ultraviolet and visible spectra were recorded on a Shumatzu 450 UV/visible spectrophotometer using quartz cells of 5 cm path length. Irradiation experiments were done as follow: 10-mL solutions were taken in a Petri dish (6 cm diameter) and irradiated with direct sunlight. The temperature was maintained at 20+2 0 C by using a constant temperature water bath. Irradiation experiments were done in Kanpur, U.P. on sunny days preferably between 9:00 and 11.00 am. The irradiance of emitted light was measured by an International Light IL-730 UV Actinic Radiometer (International Light Company, Newburyport, Mass., U.S.A.) equipped with a calibrated and cosinecorrected ultraviolet radiation detecting probe. Sunlight produced an average ultraviolet radiaton of approx. 1.2+0.1×102 erg. mm -2 . s -1 in the UV-A range (320-400 nm). It also produced approx. 1.0+0.1×10 -4 erg. mm -2 . s -1 of UV-B (285-320 nm) radiations under similar conditions. The results of intensity measurement were also confirmed by chemical actinometry 20

Results and Discussion
Singlet oxygen production A comparative study of the 1 O 2 producing ability of various dyes is summarized in Table 1. Only benzathrone was found to generate sufficient 1 O 2 to be monitored spectrophotometrically by a drop in optical density at 440 nm due to bleaching of RNO by the transannular peroxide intermediate of HIS and 1 O 2 . The 1 O 2 generating potential of benzathrone was comparable to well known photosensitizing agents like anthracene.

Dose-response relation
The formation of 1 O 2 from benzathrone was found to be both concentration and sunlight dose-dependent. A nearly linear relationship was found between the concentration of benzathrone (1-10 µg/mL) and the amount of 1 O 2 produced after 15 min exposure with sunlight ( Figure 1). The study was done using a 10 µg/mL concentration of benzanthrone. The relationship between 1 O 2 production and concentration of test compound was observed in dilute solutions only. At higher concentration the linearity was disturbed due to low solubility of benbzanthrone as well as low penetration of solar radiation into the colloidal irradiation solution.

Super oxide radical's production
It was interesting to know that metanil yellow and p-aminodiphenylamine, the two dyes which gave a negative test for 1 O 2 , were found to produce (O 2 -) in appreciable yields (Table 1 & Figure 3    ) by metanil yellow, p-ADPA and benzanthrone might rise several question about their safety particularly in conjugation with exposure to sunlight. Studies shows that, exposure to metanil yellow, p-ADPA and benzanthrone may lead to various short and long term skin photosensitization and cell damaging effects.
Photosensitized reactions of dyes leading to the formation of activated oxygen species are believed to be a matter of great concern because the later are involved in the destruction of cells, tissues, phagocytes and certain cancer inducing processes 5,7,9,11 . Moreover, Bickers et al 28  ) and other reactive forms of oxygen play a major role in the UV-induced membrane damaging reaction [29][30] .
Although spectroscopic studies of cutaneous photosensitizing agent XVII benzanthrone were carried out by Reza Dabestani et al 31 . The important roles of reactive oxygen species in diseases related to aging and the necessity and benefits of antioxidative netraceuticals in prevention of disease and promotion of healthy aging reported in the recent years 32-34 .

Conclusion
Analysis was carried out of above dyes at concentration range of (1-10 µg/mL) and exposure period. At higher concentration the linearity was disturbed due to lack of penetration of solar radiation through the colloidal system.