Dietary supplementation of vitamin C: an effective measure for protection against UV-B irradiation using fish as a model organism

Abstract

The development of UV-B protective mechanisms in aquacultural species is essential for the sustainable production of healthy aqua crop. Freshwater carp Catla catla larvae (13.5 ± 1.12 mg) were fed with a diet containing 0.5% vitamin C (D1) and a control diet (D2) for 40 days. Each group was exposed to two doses of UV-B irradiation: 360 (5 min, D1_{5 min} and D2_{5 min}) and 720 mJ cm⁻² (10 min, D1_{10 min} and D2_{10 min}) for 15 days. Significantly (p < 0.05) higher survival and average weight were recorded in D1 compared to D2 exposed to the same dose. Also, significantly (p < 0.001) higher nitric oxide synthase and lower thiobarbituric acid reactive substances and heat shock protein 70 levels were recorded in D1_{5 min} compared to the other groups. A direct relationship was found between the dose of UV-B and DNA fragmentation in muscles. DNA damage indices such as tail DNA, tail extent moment and olive tail moment were significantly (p < 0.01) lower in D1_{5 min}. Thus, supplementation of vitamin C in the diet provides UV-B protection to larvae.

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Introduction

The freshwater ecosystem is a rich source of biodiversity. Solar ultraviolet radiation B (UV-B) is an environmental stressor, which affects the freshwater environment, and the organisms that inhabit it are particularly vulnerable because they are isolated and fragmented within a terrestrial landscape.¹ The amount of ultraviolet radiation reaching the Earth's surface started increasing in the middle of the 20th century due to the deterioration of the stratospheric ozone layer.² In water, UV radiation is absorbed by dissolved organic material, but water itself hardly absorbs it.³ Hence, UV-B penetration has been observed to a depth of 55 m². In shallow rivers and streams with a low dissolved organic carbon concentration, UV-B radiation can penetrate to the bottom.^2,4 DNA damage induced by UV-B exposure is mainly due to the direct excitation of its bases, leading to mutations and chromosome fragmentation.^5,6 The alterations most frequently induced are cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimodone photoproducts (6-4PPs).⁷ These products can block DNA replication, alter gene expression by preventing transcription and cause mutagenesis or cell death.^6,8,9 The harmful effects of UV-B radiation at the organism level include increased oxidative stress, impairment in larval development, reduced movement, changes in fatty acid profiles, skin lesions, and development of cataracts and lesions to the brain.^10–13

Long term UV-B irradiation affects the immune system of carps^14–16 and rainbow trout by modulating the functions of head kidney phagocytes and lymphocytes.¹³ Exposure to UV-B radiation increases the activities of enzymes protecting against oxidative stress and also affects the molecular defence mechanism.^2,17 Some cells express protein from the family heat shock cognates 70 (Hsp 70) by a variety of environmental stress including UV-B radiation, which generates denatured proteins.^18,19 Hsp 70 plays a protective role against tissue damage by binding to denatured proteins and attempting to restore their tertiary structure and functions.^20,21

Evaluation of the response of freshwater fishes to UV-B radiation is significant since it helps in understanding the harmful effect of UV-B radiation at the organism level and simultaneously on the production of aqua crop. Catla is a eurythermal species that grows best at a water temperature ranging between 25–32 °C. They are typically surface-dwelling, zooplanktivores and usually participate in seeding migration by moving towards shallow water during monsoon season.²² However, the natural UV-B radiation is high in monsoon months and can easily penetrate the water. In Lake Naini, Delhi, India, the highest UV-B radiation (236.2 μW cm⁻²) was recorded in the month of June.²³ Also, larvae cultured in open nursery ponds receive direct exposure to sunlight, which initiates changes in their overall physiology.

Vitamin C is one of the powerful reducing agents available to cells, serving as a co-factor for the incorporation of molecular oxygen into various substrates. Dietary supplementation of vitamin C during stressful periods facilitates efficient functioning of the immune and endocrine systems.²⁴ Dietary supplementation of vitamin C also increases the regeneration ability of several fish.²⁵ Fish and crustacean cannot synthesize vitamin C since they lack the enzyme gulonolactone oxidase, which is necessary in the last step of its biosynthesis.²⁶ In the present study, we evaluated the impact of two different doses of UV-B radiation on the physiological responses of Catla catla larvae, which were used as a test model. Simultaneously, an attempt was made to confirm the ameliorating effect of dietary supplementation of the antioxidant vitamin C to reduce the harmful effects of UV-B radiation.²⁵

Materials and methods

Experimental design

Catla catla, catla larvae were collected from the Mogra fish farm, West Bengal, India and brought to the wet laboratory. Larvae were acclimated 7 days. Then the larvae (13.5 ± 1.12 mg) were divided into two groups and introduced into aquarium (15 L) following the randomised block design. The stocking density was 20 larvae per aquarium. Larvae were cultured in dechlorinated tap water and the depth of water in each aquarium was 20 cm. The larvae were fed two types of diets: the test diet containing 0.5% vitamin C (D1) and the control diet (D2) without vitamin C (Table 1) at the rate of 5% of body weight. The water temperature was 28.5 °C ± 1.5 °C, pH ranged from 7.5–7.8 and the dissolved oxygen level was 5.5 ± 0.5 mg L⁻¹. The larvae were exposed to UV-B radiation after 40 days of feeding. The impact of UV-B radiation was compared with the unexposed control in an earlier study. In the unexposed control catla larvae, their survival, growth, digestive enzyme activities and immunological parameters (lysozyme and nitric oxide synthase) were significantly (p < 0.05) higher compared to the UV-B irradiated larvae. Significantly (p < 0.05) lower carbonyl protein, thiobarbituric acid reactive substances, superoxide dismutase and heat shock protein 70 levels were found in the control unexposed catla compared to the exposed ones.^14,23 Therefore, the present investigation focused on the positive effect of dietary supplementation of vitamin C to overcome the harmful effect of UV-B in carp larvae. The larvae fed without the vitamin C supplemented diet served as the control in the present study. All experiments were performed in accordance with the guidelines and regulations of the experiments with fish, in the Department of Zoology, University of Delhi. The experimental protocols were approved by the University of Delhi.

Table 1 Ingredients used for the preparation of the test and control diets

Ingredients (g kg^−1 diet)	Test diet (D1)	Control diet (D2)
a Sea Cod (Sanofi India Limited, Mumbai, India): omega-3-PUFA, EPA and DHA. b Supradyn (Bayer Consumer Care AG, Switzerland): vitamin A = 10 000 IU, C = 150 mg, and E = 25 mg.
Dry fish powder	583.3	583.3
Wheat flour	400.2	402.7
Cod liver oil^a	10.0	10.0
Vitamin-mineral premix^b	4.0	4.0
Vitamin C	5.0	—
Proximate analysis (% dry matter basis)
Crude protein	42.1	44.63
Crude fat	6.48	7.1
Energy value (cal g^−1)	3.49	3.6

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UV-B exposure

The larvae fed with test diet (D1) and control diet (D2) were exposed to UV-B irradiation for 5 (D1_{5 min} and D2_{5 min}) and 10 (D1_{10 min} and D2_{10 min}) min daily for 15 days at a mean radiant energy of 80 μW cm⁻². The total cumulative dose of 360/720 mJ cm⁻² was received in the 5/10 min-exposed larvae during 15 days of exposure. Three replicates were used for each treatment. The individual aquarium was covered with a black plastic sheet to avoid outside light and reflectance between the aquarium itself. A Philips tube light (TL 20/12 RS, Holland) was used as the source of UV-B radiation (280–320 nm). The light was fixed at 20 cm above each aquarium. At this level the intensity of the light was 80 μW cm⁻². All the tubes were pre-burned for 100 h to give a stable output.^12,27,28 The ultraviolet radiation B at the water surface was measured using a Microtops II Sunphotometer (Solar Light Company, Philadelphia, USA). The larvae were kept under fluorescent lamps (Philips 20 W) without UV components following a photoperiod of 12 h:12 h.

Sampling

Larvae were collected and anaesthetised with MS-222 (0.01%) after 24 h of the last UV-B exposure. The survival rate and weight of each catla were recorded. Then muscle tissue sample was collected from the individual catla, pooled and stored immediately at −80 °C for various assays. For the DNA damage (Comet Assay) and heat shock protein (Hsp 70) study, the tissue was immediately processed for analysis.

Biochemical assays

Total tissue protein was determined following the method of Lowry et al.²⁹ The optical density of the sample was recorded at 750 nm using a microplate reader (Synergy HT, Biotech, USA). Tissue glutamic oxaloacetic transaminase (GOT) and tissue glutamate pyruvate transaminase (GPT) were determined using diagnostic kits (Bayer Diagnostics, Baroda, India). Absorbance was measured at 340 nm in kinetic mode using a microplate reader and levels were expressed as IU L⁻¹. Thiobarbituric acid reactive substances were measured following the method of Ohkawa et al.³⁰ Absorbance was recorded at 532 nm. The standard was prepared using 1,1,3,3-tetramethoxy propane. The result was expressed as μmol MDA per mg protein. Lysozyme activity was determined by incubating 10 μL of tissue suspension with 1 mL of Micrococcus lysodeicticus (Sigma-Aldrich, St Louis, USA) suspended in acetate buffer (0.02 M, pH 5.5) for 60 min at room temperature.³¹ The absorbance was measured at 450 nm and expressed as μg per mg tissue. The standard was prepared using hen's egg lysozyme (Sigma-Aldrich, St Louis, USA). Nitric oxide synthase was measured following the method of Lee et al.³² The absorbance was recorded at 540 nm. The nitrite concentration was expressed as mM per mg tissue. Heat shock protein 70 concentrations in muscles were quantified using the Hsp 70 ELISA kit (CUSABIO, Wuhan, China). Absorbance was recorded at 450 nm and expressed as pg mL⁻¹.

Study of DNA damage by single cell gel electrophoresis (Comet assay)

DNA damage was measured using the Comet assay method.^33,34 The slide was kept in methanol overnight and burnt to avoid contamination from machine oil and dust.³⁵ A layer of 0.1% agarose (low melting, Sigma-Aldrich, St Louis, USA) was uniformly spread on the slide (Sigma-Aldrich, St Louis) and dried at 45 °C using slide warmer (Hicon, India). This pre-coated slide was stored for future use. Tissue was minced into small pieces using a scissors in cold Hank's Blank Salt solution containing 20 mM of ethylenediaminetetraacetic acid (EDTA, Himedia, Mumbai, India) and diluted (1 : 30) with phosphate buffer saline, PBS (pH 7.4). The diluted muscle (10 μL) sample was mixed with 600 μL of 0.75% agarose. Then it was layered on the microscope slide pre-coated with 0.1% agarose.³³ The slide was kept at 4 °C for 5 min to allow the formation of agarose gel. Then the slide was incubated in lysis buffer (pH 9.5) made up of 2.5% sodium dodecyl sulphate (Himedia, Mumbai, India), 1% sodium sarcosinate (Sigma-Aldrich, St Louis) and 25 mM EDTA for 15 min at 25–30 °C. Then it was washed in distilled water for 5 min at 10 °C and electrophoresed at 2 V cm⁻¹ (Amersham Biosciences, USA) for 5 min at 10 °C. After a brief rinse in distilled water, the slide was dried at 45 °C on a slide warmer (Hicon). Then the slides was stained with propidium iodide (25 μg mL⁻¹, Sigma-Aldrich, St Louis) after brief rehydration in distilled water for 5 min. The stained slide was observed, and its image acquired using an Axio Imager Upright Microscope (2.2 Carl Zeiss, Germany). Analysis of DNA distribution in the comet was carried out with comet imager (version 2.2, Metasystem). Fifty comets from different slides were measured for each replicate. Three replicates were used for each treatment. The scored parameters including tail length, tail moment and olive tail moment were studied to quantify the DNA damage. The detailed description of the calculation was provided in the software.

Statistical analysis

Results were expressed as mean ± SEM with a significance value using the t-test statistic to compare the differences between the doses and diets. Further, to study the interaction and main effect, two-way analysis of variance (ANOVA) was conducted using the SPSS software 19.0.

Results

Survival and growth of larvae

The effect of different doses of UV-B radiation on the performance of catla larvae and the role of vitamin C in reducing the harmful effect of radiation were evaluated in the present investigation. The survival rate and average weight were significantly (p < 0.05) lower in the larvae exposed to 720 mJ cm⁻² radiation compared to that at 360 mJ cm⁻² for both feeding regimes. A significantly (p < 0.05) higher survival rate and average weight were recorded in the vitamin C supplemented diet-fed catla compared to the control group exposed to the same UV-B dose (Table 2). The two-way analysis of variance (ANOVA) of the survival and the average weight showed a significant main effect for the diet and UV-B dose, but the interaction between the UV-B and diet was insignificant (p > 0.05).

Table 2 Effect of vitamin C on the survival, average weight, tissue glutamic oxaloacetic transaminase (GOT), tissue glutamate pyruvate transaminase (GPT), thiobarbituric acid reactive substances (TBARS), lysozyme, nitric oxide synthase, heat shock protein 70 (Hsp 70) and DNA damage in catla exposed to two different doses of UV-B radiation. Data are presented as mean ± SEM

Parameters	360 mJ cm^−2 (5 min)			720 mJ cm^−2 (10 min)
	D1 diet	D2 diet	p-Value	D1 diet	D2 diet	p-Value
For DNA damage, fifty comets were studied for each replicate, and three replicates were used for each treatment (n = 50 × 3). D1 = vitamin C supplemented diet, D2 = control diet; p – value derived from statistical ‘t’ test (two-tail test); and NS = not significant.
Survival rate (%)	84.77 ± 0.15	77.5 ± 1.44	p < 0.01	80 ± 0.58	70.23 ± 0.15	p < 0.01
Average weight (mg)	257.14 ± 4.54	171.69 ± 7.25	p < 0.01	227.62 ± 6.62	139.29 ± 7.96	p < 0.01
GOT (IU L^−1)	0.11 ± 0.01	0.13 ± 0.01	NS	0.12 ± 0.002	0.14 ± 0.001	p < 0.05
GPT (IU L^−1)	0.21 ± 0.004	0.26 ± 0.001	p < 0.01	0.21 ± 0.001	0.27 ± 0.001	p < 0.01
TBARS (mmol MDA per mg tissue)	13.81 ± 1.41	77.28 ± 0.37	p < 0.01	18.93 ± 0.43	97.47 ± 0.74	p < 0.01
Lysozyme (μg per mg tissue)	148.0 ± 12.22	116.0 ± 4.0	NS	122.0 ± 10.0	86.0 ± 1.64	NS
Nitric oxide synthase (mmol per mg tissue)	73.54 ± 0.23	25.85 ± 2.71	p < 0.01	52.09 ± 3.24	24.93 ± 1.43	p < 0.01
Hsp 70 (pg ml^−1)	631.15 ± 10.88	712.03 ± 2.26	p < 0.01	772.8 ± 4.61	848.47 ± 22.14	p < 0.05
DNA damage
(a) Tail DNA	41.72 ± 1.35	52.93 ± 2.34	p < 0.01	47.77 ± 1.76	78.2 ± 3.14	p < 0.01
(b) Tail extent moment	6.36 ± 0.27	16.75 ± 1.52	p < 0.01	11.97 ± 1.16	37.79 ± 2.36	p < 0.01
(c) Olive tail moment	3.38 ± 0.13	8.62 ± 0.99	p < 0.01	5.85 ± 0.63	20.93 ± 1.37	p < 0.01

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Biochemical assays

The tissue glutamic oxaloacetic transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels were maximum in the control diet-fed larvae (D2_{10 min}) exposed to the dose of 720 mJ cm⁻² compared to the other groups. The GPT level was significantly (p < 0.05) lower in the larvae fed with the D1 diet compared to the D2 diet-fed group exposed to the same dose (Table 2). The two-way ANOVA of GOT showed an insignificant interaction as well as the main effect for the diet and UV-B dose, while GPT showed the significant main effect for the diets, but the interaction between the UV-B and diet was insignificant (p > 0.05).

The TBARS level was significantly (p < 0.01) higher in the control diet-fed larvae exposed to two different doses of UV-B radiation compared to the D1 diet-fed larvae. The TBARS level was minimum in the D1 diet-fed larvae exposed to a dose of 360 mJ cm⁻². The lysozyme level was maximum in the D1 diet-fed larvae exposed to a dose of 360 mJ cm⁻². In both feeding regimes, an inverse relationship was observed between the lysozyme level and the dose of UV-B radiation. A significantly (p < 0.01) higher level of nitric oxide synthase was detected in the D1 diet-fed larvae for both doses compared to the larvae fed with the D2 diet for the respective dose. The highest level was found in the D1_{5 min} treatment group. The Hsp 70 level was significantly (p < 0.05) higher in the D2 diet-fed larvae compared to the D1 treatment group. Hsp 70 was minimum in the D1_{5 min} group compared to the other groups (Table 2). The two-way ANOVA of TBARS and nitric oxide synthase showed significant interaction as well as the main effect for the diet and UV-B dose. Hsp 70 showed the significant main effect for UV-B dose only and significant interaction between the diet and UV-B dose (p < 0.01).

DNA damage

A direct relationship was found between the dose of UV-B radiation and DNA fragmentation (Fig. 1) in catla larvae. Significantly (p < 0.01) higher scores for tail DNA, tail extent moment and olive tail moment were found in the larvae exposed to a dose of 720 mJ cm⁻² compared to the dose of 360 mJ cm⁻² for each feeding regime. The DNA damage indices were significantly (p < 0.01) lower in the vitamin C supplemented diet-fed larvae compared to the control group (Table 2). The two-way ANOVA of tail DNA, tail extent moment and olive tail moment showed significant main and interaction effects for the diet and UV-B dose (p < 0.01).

Fig. 1 Pictures showing the DNA damage in the Catla catla larvae fed with vitamin C supplemented (D1) and control diets (D2) and exposed to either 360 or 720 mJ cm⁻² UV-B radiation. (a) D2 diet-fed larvae exposed to 360 mJ cm⁻² UV-B radiation, (b) D1 diet-fed larvae exposed to 360 mJ cm⁻² UV-B radiation, (c) D2 diet-fed larvae exposed to 720 mJ cm⁻² UV-B radiation and (d) D1 diet-fed larvae exposed to 720 mJ cm⁻² UV-B radiation. Three replicates were used for each treatment and fifty comets for each replicate (n = 50 × 3).

Discussion

Exposure to shorter UV-B wavelengths results in higher levels of oxidative stress, degrades the immune system, decreases disease resistance capacity, leads to a higher expression of heat shock proteins and induction of DNA damage in different organisms.^5,36–39 Oxidative stress results from the imbalance between the production of reactive oxygen species (ROS) principally from mitochondria and the ROS buffering activity of some enzymes or other antioxidant molecules (tocopherol, ascorbic acid and carotenes among others).^40,41 Several studies clearly showed the low survival of pelagic embryos and larvae of temperate marine fishes and tropical freshwater carp larvae exposed to UV-B radiation.^15,16,42,43 In the present study, the survival rate of catla larvae showed an inverse relationship with the dose of UV-B. Supplementation of vitamin C in their diet enhanced the survival rate of the catla larvae. Also, UV-B radiation affected the survival rate of the catla larvae.¹² Singh et al.¹⁵ reported that dietary supplementation of vitamin C and seeds of Achyranthes aspera enhanced the survival rate of UV-B irradiated Labeo rohita, rohu larvae. The lower survival rate of catla exposed to a higher dose of UV-B radiation was the result of the loss of the protective and scavenging capability of the fish against severe oxidative stress. The damage may have been formed following the direct induction of DNA damage. Also, the larvae utilize their energy for survival against radiation, which was probably manifested through slower growth rates.³⁷ A significantly higher average weight was observed in the antioxidant supplemented diet-fed catla larvae. Vitamin C is known as a booster for the immune system and detoxifying agent,^25,44 which increases resistance to several bacterial and viral pathogens in fish.⁴⁵

GOT and GPT are the indicators of liver damage. The UV-B-irradiated larvae showed a higher level of GPT in the present study. A similar result was also observed in an earlier study with catla larvae. The exposure of larvae to UV-B radiation caused higher levels of GOT and GPT compared to that in the control fish.^15,16,46 This was due to the increased lipid peroxidation by the UV-B radiation, resulting in the poor condition of the liver. Supplementation of vitamin C in the diet of the catla improved their liver function, which was evident from the reduced levels of GOT and GPT. The feeding of a higher dose of vitamin C reduced the GOT level in Japanese eel, Anguilla japonica⁴⁷ and sea bream Pagrus major.⁴⁸

The oxidation of lipids is the most commonly used approach in free radical research because many organisms, especially aquatic ones contain a high amount of lipids with polyunsaturated fatty acid residues, which are substrates for lipid oxidation.⁴⁹ The products of lipid peroxidation are unstable and categorised as primary, secondary and end products.^50,51 The most frequently used method to monitor lipid peroxidation is based on measuring the end products. Among the products, they are represented by malonic dialdehyde (MDA) and 4-hydroxynonenal.⁵² The UV-B-induced oxidative damage of the cell membranes both in vitro and in vivo were assessed by measuring the formation of lipid peroxidation products such as MDA and TBARS. All cellular components are susceptible to the action of reactive oxygen species; however, biological membranes are most severely affected.^38,53,54Girella laevifrons, sea chubs, were exposed to 1, 4 and 5 h of UV-B radiation and lipid peroxidation was maximum after 4 h exposure.²⁹ The application of HPLC and immune techniques is another approaches to measure the end products of lipid peroxidation.⁵⁵ TBARS was significantly lower in the vitamin C supplemented diet-fed larvae exposed at both doses of UV-B. Vitamin C, the major antioxidant additive used in the food industry, reduces the oxidative stress in animals.^25,48,56,57 A lower level of TBARS was found in the vitamin C supplemented diet-fed rohu larvae.¹⁵

Lysozyme activity is an important index of innate immunity in fish and is ubiquitous in its distribution among living organisms,⁵⁸ which varies depending on the degree of stress and infection. The lowest lysozyme activity was recorded in the larvae exposed to a higher dose of UV-B radiation. However, the lysozyme activity increased in the antioxidant supplemented diet-fed catla. This confirmed the role of vitamin C as first line of natural antioxidant defence against environmental stress, such as UV-B radiation.

Nitric oxide synthase activity regulates the production of nitric oxide. Nitric oxide functions as an intracellular messenger to stimulate guanylate cyclase.⁵⁹ It induces biological effects by directly activating many intercellular molecules.⁶⁰ The inducible isoform iNOS produces a large amount of nitric oxide as a defence mechanism. Feeding larvae with vitamin C supplemented diets enhances their nitric oxide synthase level. In UV-B irradiated rohu larvae, the dietary supplementation of vitamin C enhanced the nitric oxide synthase level compared the control diet-fed larvae.¹⁵

Animals can survive in adverse conditions through several ways, and one well-characterized mechanism is by the induction of stress proteins such as heat shock proteins. These proteins are synthesized constitutively in cells and are induced after exposure to stress including heat, cold, infections and diseases.⁶¹ Stress proteins are being considered for their potential role as sensitive markers of cell injury since they are related with the immune response and autoimmune diseases.⁶² In the present study, UV-B radiation induced the expression of Hsp 70. A Similar result was also observed in UV-B irradiated pike (Esox lucius) embryos, whose level of Hsp/Hsc 70 was found to be higher after 3 h of exposure.¹⁹ Many studies also reported that an increase in the level of Hsp 70 may be associated with reduced individual fitness.^63–66 This was confirmed in the present study, where a higher level of Hsp 70 was found in the control diet-fed larvae. This resulted in poor survival, growth and physiological responses in the larvae.

DNA damage can be caused directly by exposure to UV radiation or indirectly through the production of ROS.^37,67 Reactive oxygen species oxidize the bases, leading to DNA breaks.^6,68 DNA damage can lead to the expression of p53 ⁶⁹ chromosomal fragmentation⁶ and apoptosis or cellular necrosis in many organisms. Although the comet assay is a useful tool for the study of DNA damage, it has some limitations. It depends on the tissue, organism, damage causing agent, etc. Moreover, the effect on cell-cycle checkpoints is not identifiable with this assay.⁷⁰ The vitamin C supplemented diet provided efficient repair mechanisms through the antioxidant potency in the larvae. The α-tocopherol (vitamin E) present in the supplied diet (as vitamin-mineral premix) may act with vitamin C to prevent the formation of UV-B-induced CPD.⁷¹ Also, the water-soluble antioxidant vitamin C can reduce tocopheroxyl radicals directly or indirectly and thus, supports the antioxidant activity of vitamin E.⁷²

Many studies showed that vitamin C has the ability to regenerate and/or spare vitamin E in fish, such as in Atlantic salmon, Salmo salar,⁷³ yellow perch, Perca flavescens⁷⁴ and red sea bream, Pagrus major.^25,75 The results of the comet assay reflected the increase in CPD repairing processes, while there was a decrease in the p53 protein level. An earlier study showed that vitamin E inhibits the induction of CPD and oxidatively generates DNA damage.⁷¹ Hence, it reduced the DNA damage even at higher dose of UV-B radiation. A higher content of CDP dimers was recorded in UV-B irradiated Gadus morhua, Atlantic cod,³⁶ and Strongylocentrotus droebachiensis, sea urchin.³⁷ Vehniainen et al.¹⁹ reported the induction of CPDs in Northern pike exposed at the highest fluence rate of 300 and 540 mW m⁻². In sea chubs, a direct relationship was found between the duration of exposure and their tail moment.³⁹

Conclusions

The dietary supplementation of the antioxidant vitamin C provides protection against stress induced by UV-B radiation in catla larvae in culture conditions. This was evident from the significant interaction observed between the diets and UV-B doses for TBARS, nitric oxide synthase, Hsp 70 and DNA damage. Vitamin C modulated the immune system and resulted in a higher survival and average weight in the larvae. This useful information helps to provide protection to the aquacultural species in the present scenario of climate change.

Contribution of authors

RC and JGS have planned and designed the experiment. They helped MKS during experiment. MKS conducted the experiment, prepared graphs. RC has played major role in the analysis of data and manuscript preparation. PM has assisted in statistical analysis of data and related interpretation.

Conflicts of interest

The authors declare no competing financial interests.

Acknowledgements

We are grateful to Department of Biotechnology, DBT, New Delhi for financial support in the form of project to RC. KMS is thankful to the University of Delhi for providing him UGC Science Meritorious Research Fellowship.

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