Cysteine Supplementation Mitigates the Toxicity Associated with Antitumor Therapy of Ehrlich's Ascites Fluid Adsorbed over Protein A Containing Staphylococcus aureus Cowan I.

INTRODUCTION: Previously, we have reported the amelioration of ad-AF induced hepatotoxicity with the exogenous supplementation of glutathione (GSH) without compromising the anti-tumor effect of ad-AF in ascites tumor model of mice with transplantable Ehrlich's Ascites Tumor cells. Cellular uptake of glutathione (GSH) has its own limitations, therefore exogenous supplementation of L-cysteine (Cys) was tried to reduce the toxicity of ad-AF by providing -SH contents without compromising the anti-tumor property of adsorbed ascites fluid (ad-AF).
RESULTS: A significant increase in mean survival time (MST) of tumor bearing mice from 18.1 days to 32.9 days with exogenous supplementation of Cys was observed. Cys supplementation did not alter decline in body-weight gain, tumor cell counts as well as decrease in the viability of tumor cells in ascites tumor bearing animals. Similarly, Cys has been helpful to restore the hepatic -SH contents upto the levels of -SH content in tumor control group. The exogenous supplementation of Cys along with ad-AF has been helpful to restore the decline in the activities of phase-I and enhanced levels of glutathione-S-transferase (GST). The changes in the activities of different enzymes of phase-I and phase-II indicate the reduction in toxic insult induced by the therapeutic material (ad-AF). However, ad-AF treatment could not prevent tumor bearers from natural death due to tumor progression but significantly reduced the rate of tumor progression.
CONCLUSIONS: Our study suggests that exogenous supplementation of Cys alongwith ad-AF could have a potential to be developed as a modality for the treatment of ascites tumor at least at experimental level.

INTRODUCTION

Removal of circulating immune complexes (CICs) has been used as treatment strategies for various diseases both in animal models as well as in human patients.[12] The most common body fluid for the removal of CICs is blood/plasma. But in case of ascites tumor, the major source of CICs is the ascites fluid instead of sera/plasma of tumor-bearing host.[12] Earlier, we had reported the antitumor effect with the ascites fluid adsorbed over heat-attenuated and formalin-fixed Staphylococcus aureus Cowan I (SAC) using Ehrlich’s ascites tumor (EAT) model in mice. Antitumor therapy with adsorbed ascites fluid (ad-AF) was presented with hepatotoxicity.[1] Our consecutive studies have shown that an –SH-providing component such as glutathione (GSH) can mitigate ad-AF-associated toxicity without compromising the antitumor property of therapeutic material (ad-AF).[2] Removal of CICs in various diseases has been successfully performed using various ligands for the benefit of host.[123456789101112]

GSH uptake at cellular level has its own limitations, which could be a probable possibility for the failure of full recovery of –SH contents in ad-AF-treated animals.[2] Realizing these facts about GSH, in this study, we have used l-cysteine (Cys) as a source to provide –SH contents to the host. Cys is an amino acid and precursor for the synthesis of GSH. These characteristics of Cys have encouraged us to select it as an exogenous source for the recovery of the depleted –SH content in tumor-bearing animals because of the ad-AF treatment. Our data suggests that exogenous supplementation of Cys along with ad-AF treatment protected animal from the hepatotoxicity induced by ad-AF without compromising the antitumor properties of therapeutic material (ad-AF).

MATERIALS AND METHODS

Chemicals and reagents

The chemicals for enzymatic assays were purchased from Sigma Chemicals (St. Louis, Missouri, USA). The S. aureus Enrichment Broth (SAEB) was purchased from HiMedia Laboratories (Mumbai, India) and RPMI-1640 was purchased from Gibco (Billings, Montana, USA).

Animals

Animals were housed at the animal house facility of the Industrial Toxicology Research Center, Lucknow, Uttar Pradesh, India. Clearance for animal use was obtained from the Institutional Animal Ethics Committee. Eight-week-old male Swiss albino mice were obtained from the animal breeding colony of the Industrial Toxicology Research Centre. Animals were kept in plastic cages (with a daily change of sterilized rice husk bedding) and fed with pellet diet (Hindustan Lever, Mumbai, India) along with the availability of water ad libitum. Animals used for experiments were housed under controlled conditions for humidity and temperature in the animal house. Animals were acclimatized for a week to the animal house facility before the beginning of the study.

Cultivation of S. aureus Cowan I

SAC (ATCC-12598) was obtained from the American Type Culture Collection (Manassas, Virginia). Bacteria was grown in SAEB (HiMedia Laboratories) as per the methodology reported earlier.[5]

Preparation of S. aureus Cowan I suspension

SAC (ATCC-12598) was grown in SAEB for approximately 14–16 h at 37°C. The SAC cultures were harvested and washed twice with phosphate-buffered saline (PBS, ph7.4) at 2000 rpm for 10 min at 4°C. Finally, bacterial pellet was suspended to prepare a 10% (v/v) suspension with 0.5% formalin in PBS, and bacterial suspension was incubated for 3 h at room temperature with slow stirring over a magnetic stirrer. Formalin-treated bacterial suspension was extensively washed with PBS to remove formalin. Bacterial suspension (10%, v/v) was further given a heat exposure at 80°C for 5 min with slow stirring over magnetic stirrer. Heat treatment of SAC suspension was performed to heat attenuate the bacterial culture. Bacteria treated as per the aforementioned method remain stable for weeks. This 10% (v/v) bacterial suspension was further used for the removal of CICs from cell-free ascites fluid.

Expansion and maintenance of Ehrlich’s ascites tumor

Transplantable EAT was procured from the National Institute of Virology, Pune, Maharashtra, India. Tumor cell line was maintained in vivo in the peritoneal cavity of Swiss albino mice by serial transplantation. For experimental work, 1 × 106 viable tumor cells were injected intraperitoneally (i.p.) to the animals in 0.5 mL RPMI-1640.[12]

Collection of Ehrlich’s ascites fluid

The ascites fluid was collected from the peritoneal cavity of tumor-bearing mice injected with 1 × 106 viable Ehrlich’s ascites cells i.p. on 20th day post-tumor transplantation. On 20th day, sterilized 16G needle was used for the removal of ascites fluid from the peritoneal cavity of tumor bearers, which was collected in sterilized centrifuge tubes. Collected ascites fluid (with tumor cells) was centrifuged at 2000 rpm for 10 min at 4°C to separate tumor cells from the ascites fluid. Pellet of ascites cells was discarded and cell-free ascites fluid was collected after filtering with 0.22-µm filter before storing in suitable size of aliquots at -70°C.[1]

Adsorption of cell-free ascites fluid with S. aureus Cowan I suspension

Cell-free ascites fluid was mixed with 10% (v/v) SAC suspension in 1:1 ratio and incubated for 1 h at 37°C with slow stirring.[12] This bacterial suspension was centrifuged at 2000 rpm for 10 min at 4°C to pellet the bacterial mass. After centrifugation, bacterial pellet was discarded and ascites fluid was collected and stored at -70°C in suitable size of aliquots. Now ascites fluid is 1:1 diluted and designated as ad-AF for further reference. Before storing the ad-AF, it was sterile filtered with 0.22-µm filter under aseptic conditions, which was used as therapeutic material throughout this study. Reduction of CICs in ad-AF was performed using polyethylene glycol (data not shown). Treatment protocol for ad-AF is described in detail in Table 1.

Table 1

Treatment protocols for different groups of tumor-bearing animals

		Protocol for treatmenta
Group-I	Tumor control	Animals injected with ascites tumor cells and treated with 0.1 mL of normal saline i.p. on alternate days up to 15th day.
Group-II	Tumor experimental	Animals injected with ascites tumor cells and treated with 0.1 mL of adsorbed ascites fluid (ad-AF) i.p. on alternate days up to 15th day.
Group-III	Tumor experimental + Cys	Animals injected with ascites tumor cells and treated with 0.1 mL of adsorbed ascites fluid (ad-AF) along with 0.1 mL of Cys i.p. (150 mg/kg body weight) on alternate days up to 15th day.

aAnimals in each group were injected intraperitoneally with 0.5 mL of 1 × 106 viable Ehrlich's ascites tumor (EAT) cells/mL in RPMI-1640 on day 0. The animals were randomly divided into three groups having 15 animals in each group. Adsorbed ascites fluid (ad-AF) and l-cysteine (Cys) were given simultaneously but separately on the same day. Ten animals from each group were injected i.p. with normal saline, ad-AF, and Cys on alternate days as per the treatment of protocol for their respective groups till 100% mortality was observed

Viability and total tumor cell counts

On day 18, tumor control and ad-AF-treated tumor-bearing animals were killed by cervical dislocation, and ascites fluid was collected from the peritoneal cavity of mice with the use of 16G needle. Tumor cells were washed twice with RPMI-1640 for 10 min at 2000 rpm at 4°C. The tumor cells in ascites fluid were counted using hemocytometer, and the viability of tumor cells was ascertained by trypan blue dye exclusion test.[12]

Enzyme assays and –SH contents

On day 18, post-tumor transplantation, animals from each group were killed by cervical dislocation. Liver was removed from their abdominal cavity by surgical intervention and its weight was recorded. After the removal of liver, it was minced and homogenized at 10% (w/v) with PBS using medium setting (10 ups and down movement) of Potter Elvehjem homogenizer, (Thomas Scientific, Swedesboro, New Jersey, USA).

The liver homogenate was further subjected to subcellular fractionation at 9000 × g as per the method described by Hook et al.[13] This fraction of liver homogenate was used to estimate activities of different drug-metabolizing enzymes. Aniline hydroxylase (AH) and aminopyrine-N-demethylase (AD) activities were assayed by following the method of Mazel.[14] Glutathione S-transferase (GST) activity was estimated by the method of Habig et al.,[15] using a molar coefficient of 9.6 for CDNB (1-chloro-2,4-dinitrobenzene). Total sulfhydryl (TSH) and GSH contents were estimated by the method of Jollow et al.[16] The method of Lowry et al.[17] was used to estimate protein contents using bovine serum albumin as a standard.

Estimation of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase

We have used glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) as marker enzymes for liver injury. Their activities were measured by the method of Wooten.[18] For GOT estimation, aspartate (0.2 M) was used in the reaction mixture, whereas dl-alanine (0.2 M) was used for the estimation of GPT. Optical density of the end product was read at 510 nm.

Exogenous administration of l-cysteine

l-cysteine hydrochloride (SRL, Mumbai, India) was dissolved in normal saline at the concentrations of 37.5, 75, 150, and 300 mg/kg body weight.[19] Cys solution in normal saline was sterile filtered with 0.22-µm filter and stored in suitable size of aliquots at –20°C. Cys (0.1 mL) was injected i.p. with specified concentrations into the respective group of tumor-bearing animals. Cys injections were given simultaneously but separately on the same day along with 0.1 mL of ad-AF injections.

Treatment protocol

The animals for experiments were randomly divided into three groups (Group-I, Group-II, and Group-III) having 15 animals in each. Ten animals from each group were monitored for the survival as well as changes in the body weight gain till their death. The remaining five animals were killed on day 18 for different biochemical estimations. Treatment of animals in each group is described in detail in Table 1.

Statistical analysis

The statistical analysis of data and level of significance was determined by one-way analysis of variance test using Prism software, (GraphPad Software, La Jolla, California, USA (www.graphpad.com)).

RESULTS

Selection of l-cysteine dose

At first, we performed a trial experiment to select the effective dose of Cys to protect tumor-bearing mice from the toxicity of ad-AF. Mice were transplanted with EAT and simultaneously injected with 0.1 mL of ad-AF. At first four different concentrations of Cys (i.e., 37.5, 75, 150, or 300 mg/kg body weight) were used for ascites tumor-bearing animals. Cys was given i.p. on alternate days post-tumor transplantation (day 0). Cys-treated animals were monitored daily for survival and body weight gain (animal weight was taken on alternate day) over the entire period of the study. It was observed that 150 mg/kg body weight of Cys provided the best protection against toxicity-induced ad-AF with a significant increase for the survival as well as a significant decrease in body weight gain of tumor-bearing animals compared to other two lower doses of Cys (i.e., 37.5 and 75 mg/kg body weight). Lower doses of Cys failed to protect ad-AF-treated mice from mortality with the use of same quantity of ad-AF. The next higher dose of Cys, that is, 300 mg/kg body weight did not offer any additional advantages compared to 150 mg/kg body weight dose (data not shown). Therefore, for all further studies, we selected 150 mg/kg body weight of Cys.[19]

Change in tumor progression

A normal characteristic of ascites tumor is the excessive body weight gain with time because of tumor progression.[12] We observed 10%–15% body weight gain for the 1st week post-inoculation of tumor cells, even though the gain of body weight was significantly low (P < 0.05) in Group-II compared to Group-I on the 7th day. The differences in body weight gain among different treated groups were more pronounced on 14th day. On 14th day, gain in body weight was significantly lower (P < 0.05) in Group-II and Group-III compared to that in Group-I. Difference in body weight gain among treated and untreated was highly pronounced as hypothesized on day 21 post-tumor transplantation. The change in body weight can be felt visibly by noticing the belly size of the tumor-bearing animals. The percent gain in body weight of Group-I animal was 78%, whereas on the same day, body weight gain in Group-II was approximately 23%, whereas in Group-III body weight gain was 30%. Body weight gain was calculated with reference to the body weight of individual animal on day 0. The body weight gain in Group-II and Group-III was significantly lower (P < 0.001) compared to that in Group-I [Figure 1]. The group of animals given Cys along with ad-AF did not show any significant changes in their body weight gain compared to Group-II animals, which suggest that Cys did not affect the body weight gain among tumor-bearing animal treated with ad-AF. This observation indicates that Cys supplementation did not have any major effect on the antitumor properties of ad-AF. A rate of reduction in the body weight gain is an acceptable indicator for tumor regression as well as an indicator for the effectiveness of antitumor activity of therapeutic material (ad-AF).[12]

Figure 1

Effect of exogenous supplementation of l-cysteine (Cys) along with adsorbed ascites fluid (ad-AF) treatment on the changes in body weight gain in tumor bearers and treated mice. Animals in each group were transplanted with 1 × 106 Ehrlich’s ascites tumor cells intraperitoneally (i.p.); Group-I (tumor control) was administered 0.1 mL of normal saline, Group-II (tumor experimental) was treated with 0.1 mL of ad-AF, whereas Group-III (tumor experimental + Cys) received 0.1 mL of ad-AF and Cys (0.1 mL) i.p. on alternate days right from the day of tumor transplantation (day 0). Body weight gain of individual animals was recorded and percentage change in the body weight of individual animal was calculated from the initial body weight of animals (day 0). First group of bars represents the body weight gain on 7th day, second group of bars represents the body weight gain on 14th day, and third group of bars represents the body weight gain on 21st day. Values presented here are mean ± standard error (SE) of 10 animals in each group. *P < 0.05, **P < 0.005

Changes in mean survival time

Mean survival time (MST) for the animals in each group was calculated on the basis of survival data. MST was 32.9 days for Group-III compared to 18.1 days for Group-II and 24.1 days for Group-I. MST for Group-III was significantly higher (P < 0.005) compared to that for the other two treatment groups [Table 2]. Increased MST for Group-III compared to that for Group-II suggests the reduction in ad-AF-induced toxicity because of the exogenous supplementation of Cys. Increased MST in Group-III compared to that in Group-I is also suggestive of the fact that the exogenous supplementation of Cys along with ad-AF treatment did not affect the efficacy of antitumor properties of ad-AF because animals of Group-III did survive even longer than the animals of Group-I. However, ad-AF treatment prolonged the survival of tumor-bearing mice, but ad-AF could not protect the animals from the untimely death because of tumor growth, which is a natural and normal observation with any kind of antitumor treatment strategies.

Table 2

Effect of exogenous supplementation of L-cysteine (Cys) along with adsorbed ascites fluid (ad-AF) administration on mean survival time (MST) in ascites tumor bearers and treated animals

Group	Description	Mean survival time (in days)
I	Tumor control	24.1 ± 1.6
II	Tumor experimental	18.1 ± 1.2**
III	Tumor experimental + Cys	32.9 ± 2.1**

**P < 0.005

Animals were transplanted i.p. with 0.5 mL of 1 × 106 EAT cells/mL in RPMI-1640 and treated with 0.1 mL of normal saline in Group-I and 0.1 mL of ad-AF only in Group-II. Group-III received 0.1 mL of ad-AF as well as 0.1 mL of Cys separately but simultaneously on the same day. Mortality of individual animals was recorded to calculate the mean survival time. Values presented here are mean ± standard error (SE) of 10 animals

Changes in tumor cell counts and viability of tumor cells

Tumor cells in the peritoneal cavity of tumor-bearing animals of different groups were counted on 18th day. Tumor cells present in animals of Group-II were approximately 50% less compared to that in the animals of Group-I (95.4 × 106 vs. 190.3 × 106 cells/mL). This decrease in the counts of tumor cells in the peritoneal cavity ascites tumor bearers was statistically significant (P < 0.001). Similarly, we observed the viability of tumors cell in the peritoneal cavity of ad-AF-treated animals to be only 62.3% compared to the 93% viability of tumor cells in the tumor control group (Group-I). Viability of tumor cell in Group-II was significantly lower (P < 0.005) compared to that in Group-I. Cys administration showed nonsignificant increase in the viability of tumor cells compared to tumor cell viability in the animals of Group-II. Viability of tumor cells in Group-III was also significantly lower (P < 0.05) compared to that in Group-I. A decrease in tumor cell counts as well as decrease in the viability of tumor cell is a clear-cut indicator of the antitumor effect of ad-AF. Cys supplementation (Group-III) has shown nonsignificant alteration of tumor cell counts as well as viability of tumor cells compared to Group-II [Table 3].

Table 3

Effect of exogenous supplementation of L-cysteine (Cys) along with adsorbed ascites fluid (ad-AF) treatment on tumor cell counts and tumor cell viability in the peritoneal cavity of ascites tumor bearers and treated animals

Group	Description	Tumor cell counts (106/mL)	Tumor cell viability (%)
I	Tumor control	190.3 ± 10	93.1 ± 5.1
II	Tumor experimental	95.4 ± 9**	62.3 ± 6.1**
III	Tumor experimental + Cys	101.1 ± 10**	71.4 ± 7.1*

*P < 0.05, **P < 0.005

Animals were transplanted i.p. with 0.5 mL of 1 × 106 EAT cells/mL in RPMI-1640 and treated with 0.1 mL of normal saline in Group-I and 0.1 mL of only ad-AF was given to the animals of Group-II. Group-III received 0.1 mL of ad-AF as well as 0.1 mL of Cys separately but simultaneously on the same day. Tumor cell counts as well as viability of tumor cells were measured on day 18 by collecting tumor cells from the peritoneal cavity of animals from each group. Values presented here are mean ± standard error (SE) of five animals

BIOCHEMICAL CHANGES

Changes in hepatic –SH contents

ad-AF treatment has shown a significant depletion (P < 0.05) of total –SH (TSH) content in Group-II compared to that in Group-I [Figure 2]. Depletion of hepatic TSH because of the ad-AF treatment was not as striking as the depletion of hepatic GSH contents. Exogenous supplementation of Cys was helpful for the recovery of TSH in ad-AF-treated group, but recovery of hepatic TSH was marginal compared to the recovery of hepatic GSH. Our results do suggest that ad-AF treatment has a significant impact on the pool of hepatic GSH because ad-AF inoculation leads to the significant decline in GSH (P < 0.005) in Group-II compared to that in Group-I. Hepatic GSH content of Group-II remained only 45% of tumor control group (Group-I), but Cys supplementation has been helpful to bring hepatic GSH levels closer to the levels of tumor control group (68.3%). Still Cys supplementation could not recover hepatic GSH levels closer to tumor control group (Group-I). Group-III animals had shown a 23.3% recovery of hepatic GSH contents compared to Group-II. A failure for the full recovery of GSH content with Cys supplementation may be attributed to the partial intake of Cys by the hepatocytes. Partial recovery of hepatic GSH contents with Cys supplementation clearly showed a significant reduction in the levels of toxic insult caused by the treatment of ad-AF. A drastic decline of hepatic GSH contents with ad-AF suggests a preferential binding of different toxic metabolites already present in ad-AF with hepatic GSH. Probably, this is one of the reasons that Cys supplementation along with ad-AF provided protection against the toxicity of ad-AF. Further studies are needed to explain the mechanism for the protection against toxicity induced by ad-AF [Figure 2].

Figure 2

Effect of exogenous supplementation of l-cysteine (Cys) along with adsorbed ascites fluid (ad-AF) treatment on the alteration in total –SH (TSH) contents and glutathione (GSH) contents of the liver among tumor bearers and treated mice. Animals in each groups were transplanted with 1 × 106 EAT cells intraperitoneally (i.p.); Group-I (tumor control) was administered 0.1 mL of normal saline, Group-II (tumor experimental) received 0.1 mL of ad-AF, whereas Group-III (tumor experimental + Cys) was treated with 0.1 mL of ad-AF and Cys (0.1 mL) i.p. on alternate days right from the day of tumor transplantation (day 0). Animals were killed by cervical dislocation on day 18 post-tumor transplantation. TSH and GSH contents were measured in liver. First group of bars represent the total TSH contents, whereas second group of bars represent GSH contents in liver. Values presented here are mean ± standard error (SE) of five animals in each group. *P < 0.05, **P < 0.005

Changes in detoxifying enzymes

We studied two different phase-I biotransformation enzymes in this study. Our results suggest a significant decline (P < 0.005) in the activity of AD in Group-II compared to that in Group-I. A recovery of up to 85% for AD has been observed compared to tumor control group (Group-I) after Cys supplementation. A closer analysis of data suggests that approximately 20% recovery was observed in the activity of AD enzyme in Group-III compared to that in Group-II [Figure 3].

Figure 3

Effect of exogenous supplementation of l-cysteine (Cys) along with adsorbed ascites fluid (ad-AF) treatment on the alterations in the activities of phase-I and phase-II biotransformation enzymes in the liver of tumor bearer and treated mice. Animals in each group were transplanted with 1 × 106 Ehrlich’s ascites tumor cells intraperitoneally (i.p.); Group-I (tumor control) was administered 0.1 mL of normal saline, Group-II (tumor experimental) received 0.1 mL of ad-AF, whereas Group-III (tumor experimental + Cys) was treated with 0.1 mL of ad-AF and Cys (0.1 mL) i.p. on alternate days right from the day of tumor transplantation (day 0). Animals were killed by cervical dislocation on day 18 post-tumor transplantation. Activities of biotransformation enzymes were measured in liver. First group of bars represent the activity of aminopyrine-N-demethylase (AD), second group of bars represent the activity of aniline hydroxylase (AH), whereas the third group of bars represent the activity of glutathione S-transferase (GST). Values are mean ± standard error (SE) of five animals in each group. *P < 0.05, **P < 0.005

AH is another phase-I biotransformation enzyme included in this study. We have noticed that ad-AF treatment brought the activity of AH to only 67% of tumor control group (Group-I). It means that the activity of AH has been presented with a decrease of 37% in the animals of Group-II compared to the animals of Group-I. Cys supplementation has shown a significant improvement toward the activity of AH enzyme. Cys supplementation was able to show the activity of AH enzyme to 80% compared to that in Group-I. The conclusion is that Cys supplementation has shown 13% recovery of AH enzyme compared to that in tumor control group (Group-II) [Figure 3]. Changes in the activity of AD and AH enzymes suggest a strong possibility of the presence of toxicants in therapeutic material (i.e., ad-AF). Toxicants present in ad-AF could not be fully detoxified by the hepatic drug–metabolizing enzymes.

GST is a phase-II biotransformation enzyme and responsible for the catalytic reactions with endogenous and exogenous compounds, which are electrophilic in nature. Approximately 35% increase in GST (P < 0.001) activity was observed in the animals of Group-II compared to that in the animals of Group-I. Cys supplementation is again helpful to the host to rescue tumor bearers from the toxic injury induced by various toxic biomolecules already present in therapeutic material (ad-AF). Cys supplementation brought the activity of GST from approximately 135% (Group-II) to approximately 118% (Group-III), which is a significant reduction of GST activity (P < 0.05) [Figure 3]. A pattern of activities of different drug-metabolizing enzyme in this study suggests the possibilities for the presence of toxicants in therapeutic material (ad-AF). Alteration of different drug-metabolizing enzymes because of ad-AF can be brought close to the levels of enzymes in tumor control group (Group-I) by the exogenous supplementation of Cys. However, exogenous supplementation of Cys did not bring the levels of these biotransformation enzymes to the levels present in normal animal similar to the exogenous supplementation of GSH.[2]

Changes in the activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase enzymes

Activity of GOT and GPT was also measured in liver homogenate (10%, w/v) rather than sera/plasma of the tumor-bearing animals. Blood or plasma was not selected for this experiment as the availability of blood or plasma was very limited because of the anemic conditions of tumor-bearing animals. We believe that the pattern of activity of GOT and GPT will be similar to the results from plasma or serum except possibilities of differences in the absolute values of GOT and GPT in liver compared to serum/plasma. Of GOT and GPT enzymes, it was noticed that GPT is more vulnerable to the toxic insult caused by ad-AF treatment. GPT activity was only 45% in Group-II compared to that in tumor control group (Group-I). But Cys supplementation along with the ad-AF treatment showed a significant recovery of GPT activity. Cys supplementation has shown a better recovery of the GPT activity because GPT was 68.3% in Group-III compared to that in tumor control group (Group-I), which means Cys supplementation has shown more than 23% of recovery of GPT activity [Figure 4].

Figure 4

Effect of exogenous supplementation of l-cysteine (Cys) along with adsorbed ascites fluid (ad-AF) treatment on the alteration of the activities of hepatic glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) enzymes of tumor bearers and treated mice. Animals in each group were transplanted with 1 × 106 Ehrlich’s ascites tumor cells intraperitoneally (i.p.); Group-I (tumor control) was administered 0.1 mL of normal saline, Group-II (tumor experimental) was treated with 0.1 mL of ad-AF), whereas Group-III (tumor experimental + Cys) received 0.1 mL of ad-AF and Cys (0.1 mL) i.p. on alternate days right from the day of tumor transplantation (day 0). Animals were killed by cervical dislocation on day 18 post-tumor transplantation. Activities of GOT and GPT in liver homogenate were measured. First group of bars represent the GOT activity, whereas second group of bars represent GPT activity. Values presented here are mean ± standard error (SE) of five animals in each group. *P < 0.05, **P < 0.005

Group-II presented with 78.4% of GOT activity compared to the activity of GOT in Group-I. Supplementation of Cys has brought the GOT activity up to >86% in Group-III compared to that in Group-I. Therefore, supplementation of Cys has shown >8% recovery of GOT compared to that in Group-II. A recovery in GOT and GPT is indicative of improvement from the hepatic insult induced by ad-AF. Recovery of these enzymes could possibly be due to the supplementation of –SH contents using Cys, which could neutralize the toxicant already present in therapeutic material (ad-AF). Therefore, toxic biomolecules present in therapeutic material were neutralized by the exogenous supplementation of Cys, and these toxic biomolecules could not cause a significant damage to liver.

DISCUSSION

Previously, a potent antitumor response was reported with the inoculation of cell-free ascites fluid adsorbed over Protein A (PA)-containing SAC in EAT model in mice.[1] The therapeutic material (i.e., ad-AF) presented with hepatic toxicity to the host. Animals treated with ad-AF showed a significant reduction in the hepatic GSH contents as well as a decrease in the activities of different phase-I biotransformation enzymes (viz., AD and AH) along with a significant increase in the GST enzyme activity. These changes in the activities of different drug-metabolizing enzymes were suggestive of the presence of toxic components in therapeutic material, which led to the depletion of hepatic GSH pool with the inoculation of ad-AF.[1] Presentation of toxicity by antitumor drugs is neither a new nor an abnormal phenomenon. Various antitumor drugs, such as cyclophosphamide and cisplatin, have shown toxicity when these drugs are used for the treatment of cancer. Some other drugs, such as cyclophosphamide[2021] and acetaminophen,[2223] are known to produce toxic metabolites that are electrophilic in nature. Apart from drugs, some other chemicals such as butylated hydroxyanisole[23] and carcinogens such as aflatoxin[24] are also known to produce toxic metabolites that are electrophilic in nature. Presence of such toxic metabolites has been reported in ascites fluid.[25262728] The binding of toxic metabolites of electrophilic nature with GSH results in the depletion of cytosolic GSH. GSH serves as a nucleophilic substrate for the neutralization of electrophilic toxicants or toxic metabolites. Cytosolic GSH may serve as a major nucleophilic substrate for the neutralization of electrophilic toxic metabolites.[29] Increase in conjugate formation of toxic metabolites supports the faster elimination of toxic metabolites from the circulation of host. Faster elimination of toxic conjugate is helpful to minimize the insult caused by the presence or production of toxic metabolites. Overwhelming production of toxic metabolites may promote the binding of toxic metabolites to the cytosolic GSH available in the vicinity of toxicant. Binding of toxic metabolites to cytosolic GSH pool causes the depletion of cytosolic GSH.[29] If the level of GSH is low, the replenishment of –SH contents by exogenous sources may be helpful to protect the host from the toxic insult. Most of the time, liver is the major organ involved in metabolism; therefore, it is obviously the main target organ to be affected by the presence of toxicants, resulting in hepatotoxicity. The data presented in this report have clearly suggested the possibilities for the presence of toxic metabolites in ad-AF as have been previously reported.[25262728] Toxicants present in ad-AF may be the major contributing factor toward hepatotoxicity induced by ad-AF treatment.

Depletion of GSH by various toxic metabolites from drug metabolism of (viz., cyclophosphamide[2021] and acetaminophen[2223]) and chemicals (such as butylated hydoxyanisole[23]) as well as carcinogens (such as aflatoxin[24]) can be replenished with the exogenous supplementation of components, which are known to provide –SH group (e.g., GSH, N-acetyl cysteine, and Cys). Mechanistically, Cys acts as a precursor for the synthesis of GSH. Glutathione has been successfully tried to reduce the toxicity of different drugs and chemicals in vivo and even to reduce the toxicity of carcinogens such as mycotoxin.[24]

We have also reported the abrogation of toxicity induced by ad-AF with the exogenous supplementation of GSH. We have found that the exogenous supplementation of GSH did not compromise the antitumor properties of ad-AF.[2] Uptake of GSH by hepatocytes is still debatable because it requires cell membrane enzymes for the entry.[29] This was the rationale considered to select Cys, a precursor for GSH to serve as a source for –SH group to replenish the depleted levels of GSH, which may be helpful to abrogate the toxicity induced by ad-AF. Cys can be easily used as a precursor for the synthesis of GSH so that the depleted GSH can be replenished in host to neutralize the effect of electrophilic toxicants. At higher concentrations, Cys is known to undergo autoxidation and to produce thiyl radicals.[3031] Production of thiyl radicals by higher concentration of Cys has been the main reason for us to avoid the use of higher concentration of Cys in this study to ameliorate the toxic effect induced by ad-AF.

A significant drop in the activities of AD and AH enzymes was observed in this study. AD and AH was included in this study as marker enzymes to evaluate the biotransformation levels. Earlier, we had reported that AD and AH enzymes could be used as indicators for the enhanced biotransformation with the use of PA to protect against the toxicity induced by CCl4, cyclophosphamide, and endotoxin.[323334]

Ascites fluid has been reported to contain various toxic components, namely, uric acid, pseudouridine, and 1-methyladenosine.[2528] A possibility of these toxicants to induce hepatotoxicity cannot be denied, but a direct correlation of concentrations of these toxic biomolecules with tumor progression has also been established. An increase in the quantity of toxicants during adsorption of sera or plasma over SAC has also been reported.[35] Patients undergoing therapeutic intervention with the SAC-adsorbed sera/plasma have been reported to show toxic symptoms similar to the symptoms of enterotoxin toxicity. Some of these common symptoms of enterotoxin toxicity are nausea, chills, fever, and so on. SAC produces more than 20 staphylococcal toxins apart from enterotoxin.[36] Increased activities of GST also suggest the presence of toxic metabolites in therapeutic material (ad-AF) and their toxic effect on liver. The exogenous administration of Cys decreased the activity of GST (P < 0.05) in the animals of Group III compared to that of Group II. The toxicity can also be confirmed with an increase in the activities of GOT and GPT enzyme, which could be reduced with the supplementation of Cys.

Data concluded from this study suggest that supplementation of Cys along with ad-AF treatment of EAT-bearing mice protects the animals from hepatotoxicity caused by ad-AF without significantly affecting/altering its therapeutic potential. Supplementation of Cys along with ad-AF has prolonged the survival of tumor-bearing animals along with the slower rate of tumor progression. However, Cys failed to protect animals from mortality caused by tumor progression, which is just a normal phenomenon for any cancer treatment interventions. Our data suggest that further studies on the mechanism of antitumor effects and the abrogation of toxicity is the need of the hour so that this can become a useful therapy for ascites tumor patients at least on an experimental level to start with.

Financial support and sponsorship

Financial support for this study was provided by the Council of Scientific and Industrial Research (CSIR), New Delhi, India.

Conflicts of interest

There are no conflicts of interest.