Electronic ISSN 2287-0237

VOLUME

EVALUATION OF LIVER AND KIDNEY TOXICITY IN RATS RECEIVING PROTEOGLYCANS FROM FISH CARTILAGE FOR THE ACCELERATION OF BURN WOUND HEALING

FEBRUARY 2016 - VOL.11 | ORIGINAL ARTICLE

A burn wound is a type of skin injury caused by tissue damage after exposure to heat, chemicals, electrical, or radiation.A burn is one of the most common types of wound found in patients. There are up to 250,000 burn patients each year. Burns can occur in all age groups, from babies to the elderly, and are a problem in both developed and developing countries.  The  major causes of burn injury are classified as follows: flame exposure (55%), hot water exposure (scalds; 40%), and chemical and electrical exposure (5%). The incidence of burn injury is classified by age as follows: 16-64 years old (60%), 1-4 years old (20%), 5-14 years old (10%), and over 65 years old (10%).1 The severity of burn injury depends on the percentage of the total body surface area damage (%TBSA) and depths of skin layer damage (degree of burn depth). It has been indicated that more than 30% of total body surface area damage could influence a change in physical systems such as cardiovascular, respiratory, and immune systems.

Current treatment of burn wound depends on the severity of the burn: for example, drug therapy or surgery is applied to severe burn cases, but new approaches such as amniotic membrane, cytokine and gene therapy are increasingly used.1-4 Medication is the most common and easiest way to treat burns, and 1% of silver sulfadiazine (SSD) cream is used as a gold standard for the treatment of burn wounds. When SSD is exposed to the wound, it will ionize to Ag+ and inhibit the enzymes involved in the electron transport chain and bacterial DNA replication process, resulting in the killing of bacteria. SSD is absorbed through the skin to the blood vessels and then transported to liver, where metabolism occurs and more than 50% is eliminated via the kidney system in the original chemical form.5 A previous study showed that Ag+ of SSD is toxic to bone marrow, which leads to abnormal blood synthesis5 and also affects liver and kidney.6,7 Thus, the use of this drug in the patients with hepatic or renal impairment should be avoided.6-8

PG is a hybrid molecule composed of a central core protein by bonding it with polysaccharides (glycosamino- glycans or GAGs) with a covalent bond.9 PG is the major component of cartilage, and makes up approximately 90% of dry weight.10-12 It has been found that PG plays an important role in the  wound-healing  process.2,13-14  Neelam et al.13 studied the effect of PG extract of fish cartilage   on L929 fibroblast cells. The results showed that PG extract promoted fibroblast cell activation by increasing cell proliferation and migration and increasing collagen synthesis, which is involved in wound healing13 but this study is not an evaluation of toxicity. Bunman et al.2 recently studied PG extract from fish cartilage for the acceleration of burn wound healing. The results suggested that PG extracted from fish cartilage can accelerate and facilitate wound healing in rats. The combination of 1% SSD and 1% or 2% PG seem to have high efficacy in accelerating and facilitating wound healing in rats. Further investigation of the safety profile of PG and the combination of 1% SSD and PG in detail is required.2

The aim of this study is to evaluate the toxicity of liver and kidney function in rats receiving PG from fish cartilage for the acceleration of burn wound healing in an in vivo model.

Drugs and chemicals: PG solution (Garguar Lab, Co., Ltd., Thailand), SSD powder (Sigma, USA.), Sodium   pentobarbital (Nembutal; Tariqbrian Ltd, USA.).

Animals: Male Wistar (age 8 weeks, weighing 250-300 gram) were purchased from the National Laboratory Animal Centre, Mahidol University, Salaya, Thailand. The animals were housed in the Laboratory Animal Unit of the Faculty of Pharmaceutical Sciences, Chulalongkorn University under standard  conditions  of  temperature  25 ± 2 °C, 50 - 60 % humidity, and a 12 hours/12 hours light/dark cycle. The rats were kept under laboratory conditions for one week prior to the start of the experiments and allowed food and water ad libitum. At the end of each experiment, the animals were sacrificed with carbon dioxide asphyxiation. Animal experiments in this study were carried out in accordance with the Ethical Principles and Guidelines for the Use of Animals for Scientific Purposes of the National Research Council of Thailand. The animal use protocol was approved by the Institutional Animal Care and Use Committee of the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand (Protocol Approval No. 13-33-011).

Cream base preparation: Stearic acid, glyceryl mono- stearate, isopropyl myristate, sodium lauryl sulfate, glycerin,  triethanolamine, uniphen P-23 and germaben II-E were dissolved in warm water and then mixed with other ingre- dients during the cream-forming process.

SSD and PG cream preparation: SSD powder, PG solution, stearic acid, glyceryl monostearate, isopropy l myristate, sodium lauryl sulfate, glycerin, triethanol-amine, uniphen P-23 and germaben II-E were used to formulate PG, SSD or SSD+PG creams. SSD powder and/or PG solution were dissolved in warm water and then mixed with other ingredients during the cream-forming process.

Electrical hotplate inducing second-degree burn wounds: The method to induce burn wounds was inves- tigated using the method of Somboonwong,15 which was modified from Zawacki.16 Rats were randomly divided into 6 groups of 10 rats each: control rats were treated with cream base; positive controls  were  treated  with  1% SSD cream; and 4 treatment groups received 1% PG cream, 2% PG cream, 1% SSD  1%  PG  cream,  and 1% SSD + 2% PG cream. Rats were anesthetized by intraperitoneal injection with sodium pentobarbital (60 miligrams per kilograms body weight). The hair on the back was shaved. Second-degree burn wounds were induced by placing an electrical hot plate, of a diameter  of 2 cm, set at a temperature of 90 °C on a selected skin area of the back for 10 seconds. All wounds were cleaned and treated with 1 gram of cream base, 1% SSD cream, 1% PG cream, 2% PG cream, 1% SSD + 1% PG cream, or 1% SSD + 2% PG cream once daily and covered with sterile gauzes.

Evaluation toxicity

Blood collection and Preparation of plasma: Collect 0.5 ml of blood from lateral tail vein at 5 time points; baseline, on day 7,  14,  21,  and 28.17,18  The whole blood  in the test tube was  centrifuged  at  3,000  revolutions  per minute (rpm) for 10 minutes. The whole blood was separated into two layers: the upper layer which contains plasma (approximately 55% of whole blood) and the lower layer which contains erythrocytes (approximately 45% of whole blood). Plasma was stored at 2-8 oC and used within 24 hours for further analysis.19, 20

Evaluation of liver function tests: The liver function test of rats was evaluated by measuring three hepatic enzyme levels including aspartate aminotransferase, alanine transaminase and alkaline phosphatase. Twenty microliters of plasma were used for measurement of each enzyme level. The activities of aspartate aminotransferase, alanine transaminase and alkaline phosphatase were determined using assay kits which are commercially available diagnostic laboratory tests and light a ultraviolet (UV) spectrophotometer (I lab 150, Instrumentation laboratory, Italy).

Evaluation of renal function tests: The renal func- tion test of rats was evaluated by measuring levels of blood urea nitrogen and creatinine. Twenty microliters of plasma were used for measurement of each parameter. The concentrations of blood urea nitrogen and creatinine were determined using the assay kits and a UV spectrophotometer (I lab 150, Instrumentation laboratory, Italy).

Statistical analysis

Results are expressed as means±SD. Data were analyzed using one-way analysis of variance (ANOVA), followed by a Bonferroni post hoc test using SPSS for Windows, ver. 17. Values of p < 0.05 were considered to be significant.

The level of aspartate aminotransferase enzyme: On day 7, 14 and 28 post-burn the levels of aspartate aminotransferase enzyme of rats treated with 1% SSD,  1% PG, 2% PG, combination of 1% SSD + 1% PG and combination of 1% SSD + 2% PG were not significantly different when compared to the control group. On day 21 post-burn, the levels of aspartate aminotransferase enzyme of rats treated with 1% SSD, 1% PG, combination of 1% SSD + 1% PG and combination of 1% SSD + 2% PG were not significantly different when compared to levels in the control group but were significantly lower than their own baselines (p < 0.001, p < 0.001, p < 0.01 and p < 0.001, respectively) (Table 1).

Table 1: The level of aspartate aminotransferase enzyme on day 7, 14, 21 and 28 post-burn.


The level of alanine transaminase enzyme: On day 14 post-burn, the level of alanine transaminase enzyme  of rats treated with 2% PG was significantly higher (p < 0.05) than the baseline. However, on day 7, 21 and 28 post- burn, the levels of alanine transaminase enzyme of rats treated with 1% SSD, 1% PG, 2% PG, combination of 1% SSD + 1% PG and combination of 1% SSD + 2% PG were not significantly different when compared to the control group (Table 2).

Table 2: The level of alanine transaminase enzyme on day 7, 14, 21 and 28 post-burn.

The level of alkaline phosphatase enzyme: On day 7 post-burn, the levels  of alkaline phosphatase enzyme  of rats treated with 1% SSD, 1% PG, combination of 1% SSD + 1% PG and combination of 1% SSD + 2% PG were not significantly different when compared to the control group but the levels of alkaline phosphatase enzyme of all groups were significantly higher than their own baselines (p < 0.01, p < 0.001, p < 0.001, p < 0.001 and p < 0.01, respectively). On day 14 post-burn, the levels of alkaline phosphatase enzyme of rats treated with 1% SSD, 1% PG, combination of 1% SSD + 1% PG and combination of 1% SSD + 2% PG were significantly higher compared to the control group (p < 0.05, p < 0.05, p < 0.05 and p < 0.001, respectively). Moreover, the level of alkaline phosphatase enzyme of rats treated with combination of 1% SSD + 2% PG cream was significantly higher than baseline (p < 0.05). On day 21 post-burn, the level of alkaline phosphatase enzyme of rats treated with cream base and combination of 1% SSD + 2% PG were significantly higher than their baselines (p < 0.001 and p < 0.05, respectively).

However, on day 28 post-burn, the levels of alkaline phosphatase enzyme of rats treated with 1% SSD, 1% PG, 2% PG, combination of 1% SSD + 1% PG and combina- tion of 1% SSD + 2% PG were not significantly different when compared to the control group (Table 3).

Table 3: The level of alkaline phosphatase enzyme on day 7, 14, 21 and 28 post-burn.

Blood urea nitrogen: On day 7 post-burn, the levels of blood urea nitrogen of rats treated with 1% PG and combi- nation of 1% SSD + 2% PG were significantly higher than the control group (p < 0.05 and p < 0.01, respectively). Moreover, on day 7 and day 14 post-burn, the level of blood urea nitrogen of rats treated with combination of 1% SSD + 2% PG were also significantly (p < 0.05) higher than 1% SSD. On day 21 post-burn, the levels of blood urea nitrogen of rats treated with cream base and 1% PG were significantly higher than their own baselines (p0.01 and p < 0.01, respectively) (Table 4).

Table 4: The level of blood urea nitrogen on day 7, 14, 21 and 28 post-burn.

Creatinine: On day 7 post-burn, the levels of creatinine of rats treated with 1% PG and combination of 1% SSD + 1% PG were significantly higher than the control group (p < 0.05 and p < 0.05, respectively). However, on day 14, 21 and 28 post-burn, the levels of creatinine of rats treated with 1% SSD, 1% PG, 2% PG, combination of 1% SSD + 1% PG and combination of 1% SSD + 2% PG were not sig- nificantly different compared to the control group (Table 5).

Table 5: The level of creatinine on day 7, 14, 21 and 28 post-burn.

In addition, the liver enzymes including aspartate aminotransferase and alanine transaminase in the blood increase when the liver is damaged, injured or inflamed from alcohol or drugs. Aspartate aminotransferase has also been found in other cells besides liver cells such as cardiac cells and striated muscle cells. Thus, the level    of this enzyme is not specific to liver dysfunction but  can also be attributed to a disease like acute myocardial infarction. Therefore, alanine transaminase level is more specific than aspartate aminotransferase level as an indicator for the liver function test.21 On day 21, the level of aspartate aminotransferase in rats treated with 1% SSD and PG increased significantly compared to baselines but were still within the normal range (82-127 U/L).22 The level  of alanine transaminase only in rats treated with  2% PG increased significantly compared to baselines but were still within the normal range (36-64 U/L).22

Alkaline phosphatase is an enzyme in the epithelial cells of the gall bladder. This enzyme increases when the patient has an obstruction of the bile duct, small biliary tract, stones, and some liver diseases. Alkaline phosphatase is also found in other organs such as bone, placenta and intestine.23 On day 7, the level of alkaline phosphatase in rats treated with cream base increased significantly compared to baselines which may be due to dehydration from the second degree burn. The level of alkaline phosphatase of rats treated with 1% SSD and PG also increased on day 14 and 21 post-burn and decreased to normal range (82-112 U/L) on day 28.22 The increase of all three liver enzymes levels (of less than three fold) are indicated as acceptable levels in clinical settings.24-26

Blood urea nitrogen (BUN) and creatinine are the waste products from the metabolism process which are excreted by the kidney to maintain the homeostasis. High level of BUN and creatinine indicates an impairment of renal function. The baseline levels of BUN of all groups were higher than normal levels (15.6-20.1 mg/dl) reported by Diloke B etal.22 which was probably due to different housing environment of the animals. The level of BUN  of rats treated with SSD appeared to decrease, while the BUN level of rats treated with PG appeared to increase compared to baselines on the early stages of wound healing (day  7).  On day 7 post-burn, the level  of BUN  in rats treated with 1% PG was significantly higher compared to cream base and in rats  treated  with  1% SSD + 2% PG was significantly higher compared to cream base and 1% SSD. The levels of BUN  of  all groups reached their maximum levels on day 21 or 28  and dropped back to baselines at the end stage of wound healing (day 28). The decrease in BUN levels after day  21 post-burn was in accordance with other studies. The results indicated that the increase levels of BUN may partly be due to PG.

Baseline levels of creatinine in all groups were higher than normal levels (0.37-0.47 mg/dl) reported by  Diloke et al.22 which was also probably due to a different housing environment. In the early stages of wound healing (day   7 post-burn), the levels of creatinine in rats treated with 1% PG and combination of 1% SSD + 1% PG cream were significantly higher compared to cream base. However, the levels of creatinine of both groups decreased to baselines at the end stage of wound healing (day 28). The results indicated that the increased levels of creatinine may partly be due to PG. Altogether, these results indicated that the increase in BUN and creatinine levels  were partly due   to PG.

This study demonstrated that PG extracted from fish cartilage accelerated and facilitated wound healing as Bunmanand Neelam13 studied without causing toxic effects to the liver and kidney in rats in long term use. Further investigation of the safety profile of PG and the combination of 1% SSD, 1% PG and 2% PG in detail is required in clinical settings.