Exogenous supplementation of N-acetylcysteine Can Reduce Hepatotoxicity Induced by Ascites Fluid (Cell-Free) Adsorbed Over Protein-A-Containing Staphylococcus aureus Cowan-I Without Compromising Its Antitumor Effect.

INTRODUCTION: Hepatotoxicity along with enhanced mortality has remained a major concern during the development of antitumor therapy with the use of cell-free ascites fluid adsorbed (ad-AF) over Protein-A-containing Staphylococcus aureus Cowan I (SAC). Major issue with ad-AF inoculation is the significant depletion of hepatic glutathione (GSH). Exogenous supplementation of -SH contents to the host has offered an encouraging hope to explore the possibilities to use ad-AF as a therapeutic material due to its antitumor effects. GSH and l-cysteine have shown a promise with the recovery of -SH contents as well as the recovery of phase I and phase II biotransformation enzymes. Aforementioned observations prompted us to try other -SH donors.
MATERIALS AND METHODS: Therefore, in this study, N-acetylcysteine (NAC) was used as an exogenous source to provide -SH contents to reduce hepatotoxicity and mortality induced by ad-AF treatment.
RESULTS: Exogenous supplementation of NAC along with ad-AF treatment to ascites tumor bearers has shown a significant protection against hepatotoxicity and mortality caused by ad-AF. NAC substitution along with ad-AF has significantly enhanced the mean survival time (MST), without altering the antitumor effect of ad-AF as evident from tumor cell counts and viability. DISCUSSION: NAC supplementation has been successful to recover hepatic -SH contents along with the significant recovery of phase I and phase II biotransformation enzymes. Marker enzymes for liver injury have also given clear-cut indications for the recovery of tumor bearers from hepatotoxicity induced by ad-AF.
CONCLUSION: This study has shown that exogenous supplementation of NAC protects the host from the enhanced mortality and hepatotoxicity induced by ad-AF. These observations offer a hope to develop ad-AF as one of the probable treatment strategies for ascites tumors at least at experimental levels.

Introduction

Removal of circulating immune complexes (CICs) has been successfully tried as a treatment strategy of various diseases.[123456789101112] Ascites fluid is one of the major sources of CICs in case of ascites tumor bearers. In our earlier studies, we have shown antitumor effect following the inoculation of adsorbed cell-free ascites fluid (ad-AF) in animal model of ascites tumor. Enhanced mortality and hepatotoxicity were the major hurdles to reap the benefits of ad-AF as a therapeutic material for the treatment of ascites tumors.[12]

In this study, N-acetylcysteine (NAC) has been successfully used to replenish the depleted –SH contents in ad-AF-treated ascites tumor bearers, and further possibilities to develop this as a strategy to treat ascites tumor have been explored. GSH uptake at cellular level has been known to have its own limitations. A failure for the full recovery of –SH contents in ad-AF-treated animals could be attributed to some of these limitations of GSH for the uptake of GSH at cellular level.[13] Realizing these facts about GSH, in this study, we have used NAC as a source to replenish the depleted –SH contents to the ascites tumor-bearing host because NAC has been successful to reduce the adverse effects of overdoses of acetaminophen both in humans as well as in animals. NAC has numerous advantages to protect against acetaminophen-induced toxicity.[141516] NAC also acts as precursor for glutathione (GSH) synthesis and protects liver against acetaminophen-induced necrosis.[1718] Some of these characteristic qualities of NAC have encouraged us to use it as an exogenous source for the recovery of depleted –SH contents in the liver of ad-AF-treated tumor-bearing animals. This study attempted to establish that exogenous supplementation of NAC along with ad-AF treatment protects animals from the hepatotoxicity induced by ad-AF without compromising the antitumor properties of therapeutic material (ad-AF).

Materials and Methods

Chemicals and reagents

Chemicals used for enzymatic assays were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI-1640 media was purchased from Gibco (Billing, MT). Staphylococcus aureus Enrichment Broth (SAEB) was procured from HiMedia Ltd (Mumbai, India).

Animals

Animals used during these experiments were housed at animal house facility of Industrial Toxicology Research Center (ITRC), Lucknow, India. Clearance for animal use was taken from Institutional Animal Ethics Committee. Six to 8-week-old male Swiss albino mice were obtained from animal breeding colony of ITRC, Lucknow, India. Animals were kept in plastic cages (with daily change of sterilized rice husk bedding) and fed with pellet diet (Hindustan Lever Ltd, Mumbai, India). Animals were provided with food and water ad libitum. Animals were acclimatized for a week to the animal house facility prior to use for the experiments. Animal house was equipped to provide controlled conditions for the temperature and humidity to the animals housed in the facility.

Cultivation of Staphylococcus aureus Cowan I

Staphylococcus aureus Cowan I (SAC; ATCC-12598) was obtained from American Type Culture Collection (Manassas, VA). Bacteria were grown in SAEB (HiMedia Laboratories Ltd) as per the methodology described earlier.[1213]

Preparation of Staphylococcus aureus Cowan I suspension

SAC (ATCC-12598) was grown in SAEB for ~14–16 hours at 37°C with continuous slow shaking. The SAC cultures were harvested by centrifugation, and bacterial pellet was washed twice with phosphate buffered saline (PBS, pH 7.2) at 2000rpm for 10 minutes at 4°C. Bacterial pellet was suspended in PBS with 0.5% formalin to prepare a 10% (v/v) bacterial suspension. This bacterial suspension (10%, v/v) was incubated for 3 hours at room temperature with slow stirring over a magnetic stirrer. Formalin-treated bacteria were extensively washed with PBS to remove formalin. Bacterial suspension (10%, v/v) in PBS was further exposed to heat for 5 minutes at 80°C with slow stirring for heat attenuation. Bacteria treated as per the aforementioned methods remained stable for weeks at −20°C. This SAC (10%, v/v) suspension was further used for the removal of CICs from ad-AF.[1213]

Expansion and maintenance of transplantable Ehrlich’s ascites tumor in mice

Transplantable Ehrlich’s ascites tumor (EAT) was procured from National Institute of Virology, Pune, India. Tumor cell-line was maintained in vivo in the peritoneal cavity of Swiss albino mice by the method of serial transplantation. For experimental work, 1×106 viable tumor cells were injected intraperitoneally (i.p.) to animals in 0.5mL RPMI-1640 (pH 7.2).[1213]

Collection of ascites fluid from tumor-bearing mice

The ascites fluid for adsorption studies was collected from the peritoneal cavity of tumor-bearing mice on 20th day post-tumor transplantation. Animals were injected i.p. with 1×106 viable EAT cells. On the 20th day, sterilized 16G needle was used for the removal of ascites fluid (with tumor cells) from the peritoneal cavity of tumor bearers. Ascites fluid was collected in sterilized centrifuge tube. Collected ascites fluid (with tumor cells) was centrifuged at 2000rpm for 10 minutes at 4°C to separate tumor cells from the ascites fluid. Pellet of ascites tumor cells was discarded and ascites fluid (cell-free) was pooled and filtered with 0.22-µm sterile filter under aseptic conditions before storing the cell-free ascites fluid (AF) in suitable size aliquots at −80°C.[1213]

Adsorption of cell-free ascites fluid (cell-free) with Staphylococcus aureus Cowan I suspension

AF was mixed with SAC (10%, v/v) suspension in 1:1 (v/v) ratio and incubated for 1 hour at 37°C with slow stirring.[1213] AF mixed with bacterial suspension was centrifuged at 2000rpm for 10 minutes at 4°C to pellet the bacterial mass. After centrifugation, bacterial pellet was discarded and ascites fluid (cell-free) was collected. At this step, cell-free ascites fluid is already diluted to 1:1 ratio and this fluid is designated as adsorbed ascites fluid (ad-AF) for further reference. Before storing, ad-AF was filtered with 0.22-µm sterile filter under aseptic conditions. This ad-AF is used as therapeutic material throughout this study. Quantitation of CICs in AF and ad-AF was performed by polyethylene glycol precipitation method.

Tumor cell counts and tumor cell viability

On day 18, tumor-bearing animals from different groups were killed by cervical dislocation. Ascites fluid was collected from the peritoneal cavity of mice with the use of 16G needle. Volume of ascites fluid collected from individual animals was recorded. Collected ascites fluid from animals of different group was centrifuged at 2000rpm for 10 minutes at 4°C to pellet tumor cells. Supernatant was discarded and separated tumor cells were washed twice with RPMI-1640 for 10 minutes at 2000rpm at 4°C. The tumor cells removed from ascites fluid were counted using hemocytometer and viability of tumor cell was ascertained by Trypan Blue Dye Exclusion test.[1213]

Preparation of liver homogenate

On day 18, post-tumor transplantation animals from each group were killed by cervical dislocation. Liver was surgically removed from the abdominal cavity of animals and liver weight was recorded. Blood clots from liver were removed using chilled PBS. Liver was minced with scissors and homogenized with 10% (w/v) with phosphate buffer (pH 7.4) using the medium setting (10 ups and downs strokes) of Potter Elvehjem homogenizer.

Enzymatic assays and assay for –SH contents

The liver homogenate was further subjected to subcellular fractionation at 9000 × g as per the methods described by Hook et al.[19] This S-9 fraction of liver homogenate was further used for the estimation of activities of different phase I and phase II drug metabolizing enzymes and –SH contents. Aniline hydroxylase (AH) and aminopyrine-N-demethylase (AD) activities were assayed by following the method of Mazel.[20] Glutathione-S-transferase (GST) activity was estimated by the method of Habig et al.[21] using 9.6 as molar coefficient for 1-chloro-2, 4-dinitrobenzene. Total sulfhydryl (TSH) and GSH contents were estimated by the method of Jollow et al.[22] Lowry’s method was used to estimate protein contents and Bovine Serum Albumin was used as a standard to extrapolate protein contents in samples.[23]

Estimation of hepatic glutamate oxaloacetate transaminase and hepatic glutamate pyruvate transaminase

Hepatic glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) have been used in this study as marker enzymes for the assessment of liver injury. GOT and GPT activities were measured by the methods of Wooten.[24] Aspartate (0.2 M) was used as a substrate in reaction mixture for GOT. DL-alanine (0.2 M) was used as a substrate in reaction mixture for the estimation of GPT. Optical density of the end product was read at 510 nm.

Exogenous administration of N-acetylcysteine

N-acetylcysteine (HiMedia Laboratories, Mumbai, India) was dissolved in normal saline at concentrations of 50, 100, 200, and 400mg/kg body weight.[25] NAC solution in normal saline was filtered with 0.22 µm sterile filter under aseptic conditions. Sterile NAC solution was stored as aliquots of suitable size at −20°C. NAC (0.1mL) was injected intraperitoneally with specified concentrations to the tumor-bearing animals of respective groups. NAC (0.1mL) and ad-AF (0.1mL) was given as i.p. injections separately but on the same day.

Treatment protocol

For the experiments, animals were randomly divided into 3 groups of 15 animals. Ten animals from each group were monitored for the survival as well as changes in body-weight gain till 100% mortality was observed in each group. Rest 5 animals from each group were sacrificed on day 18th for the estimation of different biochemical endpoints. A detailed treatment schedule of animals is described in Table 1.

Table 1

Details of ad-AF and NAC treatment to different groups of animals

EAT bearers: Treatment protocol
Group	Descriptiona	Treatment details
Group I	Tumor Control	EAT (1×106 cells) was transplanted in the peritoneal cavity of animals. Normal saline (0.1mL) was i.p. injected on alternate days till 15th day post-tumor transplantation
Group II	Tumor Experimental	EAT (1×106 cells) was transplanted in the peritoneal cavity of animals. Animals were treated with 0.1mL of adsorbed ascites fluid (ad-AF) i.p. on alternate days up to 15th day post-tumor transplantation
Group III	Tumor Experimental + NAC	EAT (1×106 cells) was transplanted in the peritoneal cavity of animals. Animals were treated with 0.1mL of adsorbed ascites fluid (ad-AF) along with 0.1mL of NAC i.p. (200mg/kg body-weight) on alternate days up to 15th day post-tumor transplantation

aAnimals in each group were transplanted with 1×106 cells (EAT) intraperitoneally on day 0. EAT was suspended in RPMI-1640 medium before injecting to the animals. After EAT transplantation, animals were randomly divided into three groups. Each group had 15 animals. The animals were i.p. injected with 0.1mL of normal saline, 0.1mL of ad-AF, and 0.1mL of NAC into their respective groups on alternate days. NAC and ad-AF injections were given separately on the same day. Ten animals from each group were observed till 100% mortality

Statistical analysis

The statistical analysis of data and level of significance was determined by one-way analysis of variance test using Prism Software.

Results

Selection of dose of N-acetylcysteine

To select the optimal dose of NAC for the protection of host from the hepatotoxicity induced by the inoculation of ad-AF, a pilot experiment was performed. For the initial experiment, different doses of NAC viz. 50, 100, 200, and 400mg/kg body-weight were tried. Specified doses of NAC (0.1mL) were given i.p. on alternate days from the day of tumor transplantation, i.e., day 0. Changes in body weight were monitored on alternate days till 100% mortality was observed in each group.[25]

Data based on the body-weight gain and mortality suggested that 50mg/kg body-weight dose of NAC was not effective at all. Dosage of 100mg/kg body-weight did not show significant improvements compared to animals treated with ad-AF only. NAC at a dose of 200mg/kg body weight proved to be most effective in conferring protection against ad-AF-induced toxicity. Next higher dose of NAC (400mg/kg body-weight) failed to provide any additional benefits in terms of either body-weight gain or mortality compared to 200mg/kg body-weight dose of NAC. On the basis of these findings from our pilot study, we have selected 200mg/kg body-weight dose of NAC for further studies (data not shown).

Changes in tumor progression

Abnormally high body-weight gain due to tumor progression is a characteristic feature of animal model for ascites tumors.[1213] We have observed 13–18% increase in body weight among different groups of animals on 7th day after the tumor transplantation. Undoubtedly, body-weight gain was not much pronounced on 7th day, which is a usual observation due to low tumor load just after tumor inoculation. However, body-weight gain in Group III was significantly (P<0.05) lower compared to Group I [Table 2].

Table 2

Percent change in body-weight gain of ascites tumor bearers treated with ad-AF along with exogenous supplementation of NAC

Group	Descriptiona	Percent change in body weight
		7th day	14th day	21st day
I	Tumor Control	18.0±1.9	36.0±4.4	81.0±6.0
II	Tumor Experimental	10.1±1.2	26.0±4.0	32.0±3.5*
III	Tumor Experimental + NAC	13.4±1.3	31.0±3.2	37.5±4.3*

aAnimals in each group were transplanted with 1×106 cells (EAT) intraperitoneally on day 0. EAT was suspended in RPMI-1640 medium before injecting to the animals. After EAT transplantation, animals were randomly divided into three groups. Each group has 10 animals. The animals were i.p. injected with 0.1mL of normal saline, 0.1mL of ad-AF, 0.1mL of NAC, and ad-AF (0.1mL) into their respective groups on alternate day. NAC and ad-AF injection were given separately on the same day. Percent changes in body weight were calculated with respect to the initial body weight of animal. Values presented here are mean ± SE of 10 animals

*P<0.005

On the 14th day, the changes in body-weight gain among all groups of animals were quite pronounced compared to the body-weight gain on 7th day. Body-weight gain in Group II and Group III was significantly lower (P<0.05) compared to Group I. This pattern of changes in body-weight gain is indicative of antitumor effect of ad-AF. Our data suggest that NAC supplementation did not significantly affect the antitumor effect of ad-AF on day 14, but body-weight gain among tumor bearers irrespective of groups was highly pronounced on day 21. The changes in body weight on the 21st day could be visibly recognized too. Percent gain in the body weight of tumor control (Group I) was 81% whereas the body-weight gain in Group II and Group III was only 32% (i.e., ad-AF) and 37.5% (i.e., ad-AF + NAC), respectively. This significant decrease in body-weight gain on 21st day is clearly an indication for the antitumor effect of ad-AF. As expected, NAC supplementation did not significantly alter the body-weight gain. But a closer analysis of data suggests a difference in percent body-weight gain was >5% between Group II and Group III. This nonsignificant increase in body-weight gain suggests that NAC might have offered some protection against toxicity to tumor cells induced by ad-AF. The antitumor effect of ad-AF is neither significantly altered nor affected by the exogenous supplementation of NAC [Table 2].

Changes in mean survival time

Mean survival time (MST) is another important end-point for the evaluation of antitumor effect of any therapeutic material or drug. We have observed MST of 23.6 days for the animals of Group I, whereas MST for the animals of Group II was only 17.4 days. The animals of Group II were dying even earlier than the animals of Group I. The early onset of death among the animals of Group II could be due to the toxicity caused by the inoculation of ad-AF. Toxicity associated with any antitumor protocols or therapy is not an unusual observation, but exogenous supplementation of NAC along with ad-AF has increased the MST up to 34.1 days in Group III. This increase of MST was significantly higher (P<0.005) in Group III compared to Group I and Group II. Group II has shown >26% (6.2 days) decline in MST compared to Group I but the increase of MST in Group III was >95% (16.7 days) compared to Group II and >44% (10.5 days) increase in MST of Group III compared to Group I. Almost double the increase of MST in Group III was observed, which could be the result of reduction of toxicity of ad-AF due to the exogenous supplementation of NAC [Figure 1].

Figure 1

Mean survival times (MSTs) of ascites tumor bearers treated with adsorbed ascites fluid (ad-AF) along with exogenous supplementation of N-acetylcysteine (NAC). Animal from each group were injected i.p. with 1×106 cells/mice with Ehrlich’s ascites tumor (EAT) cells. Day of tumor inoculation was considered as day 0. Group I (Tumor Control) was administrated 0.1mL of normal saline, Group II (Tumor Experimental) received 0.1mL of ad-AF, whereas Group III (Tumor Experimental + NAC) was treated with 0.1mL of ad-AF and NAC (0.1mL) intraperitoneally on alternate days right from the day of tumor transplantation (day 0). The injections of ad-AF and NAC were given separately on the same day from day 0 till 100% mortality was observed. Survival of animals was recorded on every day. MST was calculated on the basis of survival of the animals. Values presented here are mean ± SE of 10 animals in each group. **P<0.005

Changes in tumor cell counts and tumor cells viability

For ascites tumor, the most convenient means of monitoring is tumor cell numbers and tumor cell viability of the tumor cells removed from peritoneal cavity of tumor bearers. The total number of tumor cells in Group I vs Group II was 205.2×106 vs 101.4×106 cells/mL, i.e., Group I had more than double the number of tumor cells compared to Group II. This decrease in number of tumor cells in Group II may be due to the following three different reasons (1) antitumor effect of ad-AF, (2) toxicity of ad-AF to tumor cells as well as to the host, and (3) either the additive or synergetic effect of both these factors. This decrease in tumor cell counts was significantly lower (P<0.001) in Group II compared to Group I. Similarly the tumor cells counts in Group III was significantly (P<0.005) lower to Group I (110.6×106 vs 205.2×106), but a nonsignificant increase in tumor cell counts have been observed with NAC supplementation in Group III compared to Group II [Table 3].

Table 3

Changes in tumor cell counts and viability of tumor cells in ascites tumor bearers treated with ad-AF along with exogenous supplementation of NAC

Group	Descriptiona	Tumor cell counts (106/ml)	Tumor cells viability (%)
I	Tumor Control	205.2±10	86.4±4.1
II	Tumor Experimental	101.4±10**	66.8±7.1**
III	Tumor Experimental + NAC	110.6±11**	68.2±4.2*

aAnimals in each group were transplanted with 1×106 cells (EAT) intraperitoneally on day 0. The animals were i.p. injected with 0.1mL of normal saline, 0.1mL of ad-AF, and 0.1mL of NAC and ad-AF (0.1mL) into their respective groups on alternate days. NAC and ad-AF injections were given separately but on the same day. Values presented here are mean ± SE of five animals in each group *P<0.05, **P<0.005

A significant decline (P<0.005) in the viability of tumor cells in Group II was observed compared to Group I, which was 66.8% vs 86.4%. Exogenous supplementation of NAC along with ad-AF has shown a nonsignificant change in the viability of tumor cells in Group III compared to Group II (68.2% vs 66.8%). Although, the viability of tumor cells in Group III has remained significantly low (P<0.05) compared to Group I (68.2% vs 86.4%) [Table 3].

Biochemical changes

Changes in hepatic total sulfhydryl and hepatic glutathione contents

Depletion of both TSH contents and GSH contents in liver have simply attested the possibilities for the hepatotoxicity induced with the inoculation of ad-AF. These two observations are very important to address the concerns related with enhanced mortality due to the treatment of ad-AF. We have observed a significant (P<0.005) decline of hepatic GSH in Group II compared to Group I [Figure 2]. The values have been expressed as (100% vs 51%) in Group I vs Group II, so there was ~50% decline in hepatic GSH contents in Group II compared to Group I, which could be due to the toxicity induced by ad-AF. Exogenous supplementation of NAC along with ad-AF has protected animals from the toxic effects of ad-AF because Group III showed hepatic GSH contents as >73% compared to Group I. In other words, NAC supplementation successfully replenished >22% hepatic GSH in Group III compared to Group II; however, NAC could not replenish GSH fully to the levels of tumor control. Therefore, hepatic GSH levels in Group III were >22% higher compared to Group II, which still remained <26% compared to Group I [Figure 2].

Figure 2

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

The TSH content has also shown a significant decline (P<0.02) in Group II compared to Group I due to the treatment of ad-AF, but this decline was not as sharp as the decline of GSH following the treatment with ad-AF. The exogenous supplementation of NAC to the host did not show significant recovery of TSH compared to Group II. In summary, we can conclude that the NAC supplementation has marginal effect on the recovery of hepatic TSH. Though, at this stage we are unable to explain this observation [Figure 2].

Changes in phase I and phase II biotransformation enzymes in liver

Hepatotoxicity has turned out to be one of the major concerns to develop ad-AF as an antitumor therapy; therefore, we have included different enzymes from phase I and phase II biotransformation system of liver in this study. We have observed ~40% decline in the activity of AD in Group II compared to Group I, which was significantly low (P<0.005). The recovery of AD in Group III was never complete as compared to Group I, though exogenous supplementation of NAC has been successful to recover AD up to 87.2%. This recovery of AD in Group III was significant (P<0.001) in comparison to Group II (87.2% vs 60.1%) [Table 3].

Similar to the activity of AD, we have also observed a significant decline (P<0.001) in the activity of AH i.e., another phase I biotransformation enzyme included in this study. AH levels were >38% lower (P<0.001) in Group II compared to Group I (100% vs 60.1%). This decline in AH may be attributed to the toxicity caused by ad-AF inoculation, though exogenous supplementation of NAC has resulted in significant recovery of AH in Group III compared to Group II. NAC has recovered AD levels >21% in Group III when compared to Group II but remained >17% lower in Group III compared to Group I [Figure 3]. Decline in the activities of phase I biotransformation enzymes, i.e., AD and AH, suggests strong possibilities for the presence of toxic metabolites in the therapeutic material, i.e., ad-AF.

Figure 3

Effect of exogenous supplementation of N-acetylcysteine (NAC) along with adsorbed ascites fluid (ad-AF) treatment on the alterations of phase I and phase II biotransformation enzymes activities in liver of tumor bearers and treated mice. Animals in each groups were transplanted with 1×106 EAT cells i.p. Group I (Tumor Control) was administrated 0.1mL of normal saline, Group II (Tumor Experimental) received 0.1mL of ad-AF, whereas Group III (Tumor Experimental + NAC) was treated with 0.1mL of ad-AF and NAC (0.1mL) i.p. on alternate days right from the day of tumor transplantation (day 0). Animals were sacrificed by cervical dislocation on 18th day 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) in liver. Values are mean ± SE of five animals in each group. *P<0.05, **P<0.005

GST was selected as a phase II biotransformation marker enzyme as it plays an important role in conjugation of toxicants in the host. We have observed a significant increase (P<0.05) in GST activities in Group II compared to Group I (140.1% vs. 100%). Increased activity of GST is indicative for the possibilities of the conjugation of electrophilic toxicants present in therapeutic material, i.e., ad-AF. Exogenous supplementation of NAC has reduced the activities of GST significantly (P<0.05) in Group III compared to Group II (106.4% vs 140.1%) amounting to a recovery of >33% [Figure 3]. So the overall indications from the activities of phase I and phase II enzymes suggest either the presence of toxicants in ad-AF or toxicity induced by therapeutic material (ad-AF), which could be causing hepatotoxicity as observed in these animals.

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

Enzymes such as GOT and GPT are considered as excellent biochemical markers for liver injury, and in this study hepatotoxicity is one of the major concerns for antitumor effect of ad-AF. Instead of using serum/plasma of tumor-bearing animals, we have selected liver as the source for the estimation of GOT and GPT. The main reason for the selection of liver to estimate GOT and GPT for this study was the anemic conditions of tumor-bearing animals. Certainly, there would be a quantitative differences in the levels of hepatic GOT and GPT compared to the levels of GOT and GPT in serum/or plasma, but the pattern of activities of GOT and GPT may remain same from either sources.

We have observed that GPT is more affected by ad-AF compared to GOT. GPT was significantly reduced (P<0.005) in Group II compared to Group I, i.e., 49% vs 100%. Supplementation of NAC has shown an excellent recovery of GPT up to a level of 71.4%, so the differences in the recovery of GPT was >22%. Even though the recovery of GPT was significant, but the difference in GPT activity was >28% in Group III compared to Group I (71.4% vs 100%). This difference in GPT could be the due to the toxicity of ad-AF as well as to maintain antitumor activity of ad-AF by killing the tumor cells [Figure 4].

Figure 4

Effect of exogenous supplementation of N-acetylcysteine (NAC) 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 groups were transplanted with 1×106 EAT cells i.p. Group I (Tumor Control) was administrated 0.1mL of normal saline, Group II (Tumor Experimental) was treated with 0.1mL of ad-AF, whereas Group III (Tumor Experimental + NAC) received 0.1mL of ad-AF and NAC (0.1mL) intraperitoneally on alternate days right from the day of tumor transplantation (day 0). Animals were killed by cervical dislocation on 18th day post-tumor transplantation. Activities of GOT and GPT were measured in liver homogenate. First group of bars represent the GOT activity, whereas second group of bars represent GPT activity in liver homogenate. Values presented here are mean ± SE of five animals in each group. *P<0.05, **P<0.005

The pattern of GOT activity has also demonstrated a pattern much similar to GPT except that differences are quantitative in nature. The enzymatic levels of GOT was significantly lower (P<0.05) in Group II compared to Group I, i.e., 72.6% vs 100%. The activities of GOT has also showed significant recovery with exogenous supplementation of NAC along with ad-AF. NAC supplementation has shown >15% recovery of GOT levels in Group III compound to Group II, i.e., 88.3% vs 72.6%, but activity of GOT remained <11% in Group III compared to the activity in Group I (88.3% vs 100%) [Figure 4]. Our observation of GOT and GPT activities suggest that the hepatotoxicity induced by ad-AF was recovered significantly with the supplementation of NAC.

Discussion

CICs have been reported as one of the major contributing factors for the induction as well as maintenance of immunosuppression in tumor bearers across various animal models as well as human patients.[123456789101112] Various strategies have been successfully tried with the use of different types of ligands for the removal of CICs from the circulation of tumor bearers. The most commonly used biological fluid of choice for the removal of CICs is either blood or plasma.[3456789101112] In this study, we have used ascites fluid for CICs removal instead of sera or plasma because ascites fluid is produced in huge amount in ascites tumor bearers. Due to the fast progression of ascites tumor, a significant increase as well as accumulation of ascites fluid has been routinely observed in the peritoneal cavity of ascites tumor bearers. Ascites fluid has been reported as the major source of CICs rather than blood/or plasma at least in case of ascites tumors.[1213] SAC adsorbed ascites-fluid (ad-AF) was used as a therapeutic material for this study because (1) removal of CICs from ascites fluid could induce immune-potentiation, (2) waste body fluid (autologous) can be conveniently used for treatment, and (3) as ascites fluid is autologous in origin, reintroduction of ad-AF may not cause any major adverse immunological response. SAC has been selected for the removal of CICs from ascites fluid (cell-free) because >40,000 molecules of Protein A (PA) are present on each bacterium,[26] and PA uniquely binds more effectively with CICs compared to the binding of PA to Fc portion of IgGs.[27] The added advantage for the use of bacterial suspension is that the production of bacterial suspension is one of the most cost-effective method compared to other methods such as use of purified PA.

In this study, enhanced mortality and hepatotoxicity was observed in tumor bearers treated with ad-AF. Another prominent observation was depletion in hepatic GSH in tumor bearers after ad-AF treatment.[1213] Earlier, GSH and L-cysteine have been successfully tried to reduce the ad-AF-induced hepatotoxicity.[213] Unfortunately, either GSH or L-cysteine supplementation did not protect animals completely from mortality and hepatotoxicity caused by ad-AF. Exogenous supplementation of GSH and L-cysteine could not confer protection due to inherent limitations such as direct uptake of GSH as well as autolysis of L-cysteine.[28] Both these limitations remain possible concerns as these could be the reasons for the failure of complete recovery of animals from ad-AF induced toxicity even after the exogenous supplementation of GSH or L-cysteine.[213] These concerns and observations lead us to design this study where ad-AF treated animals were supplemented with NAC to replenish the depleted hepatic GSH contents.

NAC is considered as a precursor in the metabolic pathway of GSH synthesis.[17] NAC is a safe and inexpensive drug which is commercially available for quite some time by now.[1415161729303132] NAC is known to replenish intracellular GSH because NAC is an excellent source to supply sulfhydryl group. NAC stimulates GSH biosynthesis, to promote detoxification as well as to act as a free radicals scavenger.[17] Lately, NAC has also been successfully tried to replenish GSH in HIV infections.[33] Exogenous supplementation of NAC to ad-AF treated animals might replenish hepatic –SH content so that toxicity of ad-AF can be ameliorated. Amelioration of toxicity by NAC is attributed to its dual role of serving as a nucleophilic substrate as well as being a –SH donor.[17] The presence of thiol group makes NAC as an excellent antidote in case of acetaminophen poisoning.[17] Another added advantage is that NAC is equally effective either with oral administration or intravenous administration in reversing the hepatotoxicity caused by acetaminophen poisoning.[2930] NAC has been reported to protect normal cells from the toxicity caused by chemotherapy or by radiation therapy, too.[34] NAC has also been shown to have immunomodulatory effect during immunosuppression and oxidative stress.[35]

NAC supplementation has been tried to replenish hepatic GSH content so that NAC may reduce hepatotoxicity caused by ad-AF. The toxicity of ad-AF has been further confirmed with changes in levels of marker enzymes for liver injury as well as alterations in activities of different hepatic phase I and phase II biotransformation enzymes. NAC supplementation is helpful to bring back these enzymatic activities closer to the normal levels [Figures 2 and 3].

Certainly toxicity by antitumor drugs is neither a new nor an abnormal phenomenon. Drugs such as cyclophosphamide, cisplatin have been presented with toxicity when used for cancer treatment.[3637] These anticancer drugs produce different toxic metabolites that are electrophilic in nature. Production of electrophilic toxic metabolites have been reported from chemicals (BHT) and carcinogens such as aflatoxins.[3839] Ascites fluid itself contains various toxic biomolecules that are electrophilic in nature.[40414243] Treatment of ad-AF has resulted in hepatic GSH depletion, which indicates toward the possibilities for the presence of toxic metabolite of electrophilic nature in therapeutic material. Under optimal conditions, toxic metabolites get conjugated and removed from host by different drug metabolizing enzymes such as phase I and phase II biotransformation system of liver. We have also observed significant changes in phase I and phase II enzyme, which strongly suggest that the toxicity caused by toxic metabolites either present in therapeutic material or produced during metabolism of therapeutic materials. Majority of time, liver is the major organ for metabolism; therefore, liver could be an obvious target that would be affected by the presence of toxicants leading to hepatotoxicity. Hepatotoxicity induced by ad-AF treatment was further confirmed by the altered activity of liver injury marker enzymes, i.e., GOT and GPT [Figure 4]. Therefore, replenishment of –SH contents was tried as a strategy to either circumvent or to reduce toxicity.

Earlier, we have been successful to reduce hepatotoxicity of ad-AF without compromising antitumor effect of ad-AF with exogenous supplementation of GSH and L-cysteine.[2.13] In this study, some of the advantages associated with NAC supplementation may offer even better protection against ad-AF-induced hepatotoxicity, because NAC has been successful to reduce toxicity of acetaminophen poisoning.[14151617]

We have included AD and AH as marker enzymes to study the effect on biotransformation in this study too. Earlier we have reported AD and AH enzyme activities could be used as indicators for enhanced biotransformation as well as an indicator to protect against the toxicity induced by CCl4, cyclophosphamide, and endotoxin.[444546] Some of the toxic metabolites such as uric acid, pseudouridine, and 1-methyladenosine have been reported to be present in the ascites fluid.[404142] Possibilities of these metabolites to play a role in hepatotoxicity cannot be denied. Apart from toxicants already present in AF, a further addition of toxicants/or bacterial toxins of SAC origin in ad-AF is also a real possibility. Leaching of bacterial toxins of SAC origin has been earlier reported with the adsorption of plasma/sera of patients with SAC. Even patients undergoing therapeutic intervention with the SAC-adsorbed sera/plasma have been presented with toxic symptoms similar to the symptoms of enterotoxin/s toxicity. Some of the common symptoms of enterotoxin toxicity such as nausea, chills, fevers have been previously reported.[47] As SAC is known to produce more than 20 toxins apart from enterotoxin, all of them can together contribute toward enhanced toxicity with the use of ad-AF as a therapeutic material.[48] Increased activities of GST suggest the presence of toxic metabolites in therapeutic material (ad-AF) and their hepatotoxicity. The exogenous administration of NAC has decreased the activity of GST in the animals of Group III compared to Group II [Figure 3]. The toxicity can also be confirmed with increase in GOT and GPT activities. NAC supplementation was helpful in reducing the level of GOT and GPT enzymes in tumor bearers.[Figure 4].

This study can safely conclude that supplementation of NAC along with ad-AF treatment of EAT-bearing mice protects animals from the hepatotoxicity caused by ad-AF without significantly affecting or compromising the therapeutic potential of ad-AF. Supplementation of NAC along with ad-AF has prolonged the survival time of tumor-bearing animals and in decreasing the rates of tumor progression. However, NAC failed to protect animals from mortality resulting due to tumor progression, which is a normal phenomenon for any intervention so far tried for cancer treatment. Our data suggest that further studies on the mechanism of antitumor effects and abrogation of toxicity is the need of the hour so that ad-AF can become a useful therapy for ascites tumor patients at least at experimental level to start with.

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Conflicts of interest

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