Studies on Production and Chemical Property of Singlet Oxygen and Superoxide Radical by Dyestuffs

: There are several low lying singlet oxygen ( 1 O 2 ) and superoxide radical ( ) − 2 O which are important in photochemical oxidation. In our present analysis we are studying chemical property of singlet oxygen ( 1 O 2 ) and super oxide radical ( ) − 2 O and some dyestuffs species to produce reactive oxygen such as singlet oxygen 1 O 2 and superoxide radicals. Irradiation with sun light in vitro the dyestuff like benzanthrone, metanil yellow and p -aminodiphenylamine were found to produce reactive oxygen species such as singlet oxygen ( 1 O 2 ) and/or superoxide radicals ( ) − 2 O .The dose response relationship between singlet 1 O 2 production when sunlight expose of those dyestuffs (0-25 min), and (0-12 min) for super oxide ( ) − 2 O production were studied .However benzanthrone produces detectable amount of 1 O 2, Although metanil yellow and p -aminodiphenylamine ( p -ADPA) did not produce detectable amounts of 1 O 2 under similar conditions. The above dye stuffs are routinely used in textiles, cosmetics, detergents, leather industries as well as food additives and role of these activated oxygen species in the development of skin diseases.


Introduction
Production of synthetic dyes in India 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 ), superoxide radical (HO 2 or ( ) − 2 O ), 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-1naphthalenyl) 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 cosine -corrected ultraviolet radiation detecting probe.Sunlight produced an average ultraviolet radiation of approx.1.2+0.1×10 - erg.mm -2 .s -1 in the UV-A range (320-400 nm).It also produced approx.1.0+0.1×10 - erg.mm -2 .s - of UV-B (285-320 nm) radiations under similar conditions.The results of intensity measurement were also confirmed by chemical actinometry 20 .Formation of 1 O 2 was determined in aqueous solutions by the method of Kraljic and Mohsini 21 .With water insoluble chemicals, reactions were done in dimethyl formamide (DMF) solution (5-20% in 0.01 M phosphate buffer, pH 7).A 10 mL solution of RNO was prepared in a phosphate buffer (0.01 M, pH7) and optical density was adjusted between 1.2 and 1.4.Histidine (1×10 -2 M) was added to the RNO solution as a selective acceptor of 1 O 2 .Stock solutions (1 mg/mL) of test compounds were prepared in ethanol, DMF or dimethyl sulfoxide (DMSO).RNO and HIS samples were irradiated with various concentrations of test compounds (0.1, 1.0 and 10µ g/mL) and sunlight dose (0-25 min).Singlet oxygen forms a transannular peroxide intermediate with HIS leading to the bleaching of 1 O 2 was also established by examining the reaction kinetics in D 2 O and by carrying out quenching studies with sodium azide 22  O were obtained by performing quenching studies with SOD 26 .

Singlet oxygen production
A comparative study of the 1 O 2 producing ability of various dyes are studied.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.

Singlet oxygen 27
The lowest energy electron configuration of O 2 , which contains two electron in π orbital's, gives rise to three states is given in Table 1.The 1 ∆ g states has a much longer life time than the 1 ∑ + g states 28 .The life time is solvent dependent, varying by order of magnitude in organic solvents.There are two main ways of generating the singlet oxygen molecules: 1 photo chemically by irradiation in presence of sensitizer and 2 chemically.The photochemical route proceeds as follows, where sense represents the photo sensitizer as shown in equation from 1 to4

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

Superoxide radical's production
There are several low lying singlet states that are important in photochemical oxidation .Like NO ,which has one unpaired electron in an antibonding (π*)MO ,oxygen molecules associate only weakly ,and true electron pairing to form a symmetrical O 4 species apparently does not occur even in solid.Both liquid and solid O 2 are pale blue.The comparative study of the various bond values for dioxygen species is summarized in Table 2.

Chemical property of dioxygens
Oxygen combines with almost all other elements, usually however, only on heating.Dioxygen will some time react reversibly 30 with certain metal complexes and their ligand behavior of O 2 .Many studies have been made on the mechanism 31

of the reduction of O 2
. There is no evidence for four or two electron reduction steps ,as would be suggested by the over all reactions from equation no 5 to 9.
The oxidation of water to O 2 in photosynthetic system in plants has been greatly studied 32 as have synthetic models such as those of manganese complexes; the oxidation seems occur likely to proceed via H 2 O 2 rather than OH .Radicals and probably two or four manganese centre are involved 33.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 ( ) − 2 O in appreciable yields (Figure 2 and 3).Benzanthrone produced ( ) − 2 O in almost the same quantity as produced by anthracene and hematoporphyrin 34 (Figure 4).

Results and Discussion
Selected dyes and dyestuffs, benzanthrone, orange II, metanil yellow and p-ADPA most commonly used in colouring and textile industries as well as food additives were examined for their ability to produce activated oxygen species 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  O and other reactive forms of oxygen play a major role in the UV-induced membrane damaging reaction [36][37] .Although spectroscopic studies of cutaneous photosensitizing agent XVII, benzanthrone were carried out by Reza Dabestani et al 38 .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 recent years [39][40][41] .
Production of 1

Conclusion
Analysis was carried out of above dyes at concentration range of (1-10 µg/mL) and exposure period (0-25 min) for obtaining dose response relationship and (0-12 min) for determination of ( ) − 2 O production of above dyestuffs.At higher concentration the linearity was disturbed due to lack of penetration of solar radiation through the colloidal system.

2 O 2 O
photosensitized reduction of NBT25 with some modifications.Reduction of NBT by ( )− 2 Oleads to the formation of a blue colored product (nitro blue formazan), which is quantitatively estimated spectrophotometrically at 560 nm.A 1.67×10 -4 M solution of NBT was prepared in carbonate buffer (0.01 M, pH 8) containing 5-20% DMF to keep the reaction product in solution.10 mL solutions were irradiated with various concentrations of test samples in sunlight for varying lengths of time.Further evidences for the generation of ( ) − 2

2 O
1 O 2 produced after 25 min exposure with sunlight Figure 1.The study was done using a 10 µg/mL concentration of benzanthrone.The order of food stuffs tested aganaist chemical agent for ( ) − production are benzanthrone > metanil yellow > p-ADPA were found (as shown in Figure 2, 3 & 4 ).

Figure 1 .
Figure 1.Dose-response relationship between 1 O 2 production and sunlight exposure of benzanthrone 25 min.

Figure 4 .
Figure 4. O - 2 production by photosensitized reduction of NBT (1.6x10 -4 M in 0.01 M) carbonate buffer with 5-20% DMF, pH 8 using 10 µg/mL of benzanthrone (reaction time 12 min.) The first step is one electron reduction to the superoxide radical ion O 2 -.The potential for reduction O 2 + e = O − 2 (7) Ranges from ~ -0.2to -0.5 V depending on the medium.The O 2 -ion is a moderate reducing agent, comparable to dithionite, and a very weak oxiding agent's .Thus most of the oxidation by O 2 is due to peroxide ion H O

2 O 2 O
[ 1 O 2 and ( ) − ] under normal sunlight with a view to investigating the possible occupational health hazard to people through a series of photodynamic oxidation reactions leading to cell damage.The formation of 1 O 2 by benzanthrone and ( ) − 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.

Table 1 .
States of O 2 molecule.
29ergy transfer from triplet excited 3 sens* to 3 O 2 to give 1 O 2 is a spin allowed process .A representative example is the use of excited azoalkane with O 229.

Table 2 .
Various bond values for dioxygen species.
15Moreover, Bickers et al35have demonstrated the destruction of epidermal cytochrome P-450 in epidermal microsomes by1O 2 generated through photosensitization reactions induced by hematoporphyrin derivatives.Several other studies have also suggested that 1 O 2 , ( ) O 2 and O 2 -at different temperature Incubation of reagent mixture used for determining 1 O 2 and O 2 -in the presence of all test compounds at 0, 25 and 50 O C did not produce any 1 O 2 and O 2 -.The results were in agreement with our concepts that photo excitation is necessary for the generation of 1 O 2 and O 2 -species from test compounds under our experimental condition .