Cyclophosphamide-induced multiple organ dysfunctions: unravelling of dose dependent toxic impact on biochemistry and histology.

Crack cocaine is a very toxic product derived from cocaine. The aim of this study was to evaluate genetic damage in multiple organs of rats following acute exposure to crack cocaine. A total of 20 Wistar rats were distributed into four groups (n = 5), as follows: 0, 4.5, 9, and 18mg/kg body weight (b.w.) of crack cocaine administered by intraperitonealroute (i.p.). All animals were killed 24h after intraperitoneal (i.p.) injection. The results showed that crack cocaine increased the number of micronucleated cells in bone marrow cells exposed to 18mg/kg crack cocaine (p < 0.05). Peripheral blood and liver cells presented genetic damage as depicted by single cell gel (comet) assay at 9 and 18mg/kg doses (p < 0.05). Immunohistochemistry data revealed significant increase in 8-hydroxy-20-deoxyguanosine (8-OHdG) immunoexpression in hepatocytes of animals exposed to crack cocaine at 9 and 18mg/kg (p < 0.05) when compared with negative controls. Taken together, our results demonstrate that crack cocaine is able to induce genomic damage in multiple organs of Wistar rats.

We have found previously that daily treatment of male rats for 11 wk with low doses of the anticancer drug cyclophosphamide had no apparent effect on male reproductive organ weights, epididymal sperm counts, or serum hormones at the end of the treatment period; yet, upon breeding to untreated females, these males produced a high rate of post-implantation loss and fetal anomalies. The present study was designed to investigate the time course and dose response of the effects of chronic cyclophosphamide treatment on the male reproductive and hematologic systems. Male Sprague-Dawley rats were gavage-fed for 1, 3, 6 and 9 wk with saline (control), or 5.1 (low dose) or 6.8 (high dose) mg/kg/day of cyclophosphamide. After each of the treatment periods, males were mated to determine the effect on pregnancy outcome, then killed, and the effects on the male reproductive and hematologic systems were assessed. After 6 wk of treatment, a sharp increase in mortality was found between the 5.1 and 6.8 mg/kg/day doses of cyclophosphamide. The high dose of cyclophosphamide induced higher levels of pre- and post-implantation loss but fewer fetal anomalies than did the low dose. The low dose of cyclophosphamide did not affect reproductive organ weights; in contrast, the high dose caused decreases in epididymal, ventral prostate, and seminal vesicle weights after 3, 6, and 9 wk. Testicular and epididymal sperm counts were decreased in a dose-dependent manner after 3 wk; in addition, the high dose led to a decrease in epididymal sperm counts after 6 wk of treatment. Another rapidly proliferative tissue, the bone marrow, was dramatically affected by both doses of cyclophosphamide at all time points, with leukocyte counts decreasing to 40% of control by 1 wk. After 9 wk of treatment, effects on the male reproductive system were less marked, compared to earlier time points, whereas those on the hematologic system and pregnancy outcome persisted. Thus chronic low-dose treatment of male rats with cyclophosphamide not only had early and striking effects on the bone marrow and the pregnancy outcome but also affected the male reproductive system in a clear time- and dose-dependent manner.

Assessment of reproductive, genotoxic, and cytotoxic effects of leachate-contaminated water in male mice

A method is described for the isolation of large numbers of newborn larvae from adult Trichinella spiralis. These isolated larvae were used in experiments which tested their ability to infect mice via the intraduodenal, intravenous, and intraperitoneal routes, and to infect rats via the intraperitoneal and intravenous routes. Encysted muscle larvae were seen in diaphragm preparations from mice injected intravenously and intraperitoneally 14 days previously with newborn larvae. No muscle larvae were seen in diaphragm muscle preparations from mice which had received newborn larvae intraduodenally. Quantitative studies on larval infectivity were carried out in rats. Larval counts showed that 66% of the larvae were recoverable as mature muscle larvae from rats injected iv, while only 2% of the original dose was recovered from ip injected rats. In another experiment using larger numbers of rats in each group, newborn larvae were 73% infective in iv injected rats, and only 9% infective in ip injected rats. Nembutal, administered ip to rats prior to infection with newborn larvae, had no major effect on larval infectivity. Limited numbers of newborn larvae have been isolated from host body cavities (Matoff, 1940; Berntzen, 1965; Shanta and Meerovitch, 1967) and blood (Matoff, 1943b; Gould et al., 1955; Phillipson and Kershaw, 1961) during early phases of intestinal infection and have been described in morphological detail (Richels, 1955; Ali Khan, 1966). Denham (1967) collected parenterally infective newborn larvae after incubation of gravid females for 24 to 48 hr. However, isolation of large numbers of newborn larvae of a determinable age has not previously been described, although Larsh (1963) and others have suggested that this would be necessary in order to better correlate the stage of the parasite with host immunity. Inoculation of gravid female worms and newborn larvae of Trichinella spiralis demonstrated that newborn larvae could infect dogs and rats by the parenteral route (Matoff, 1943a). However, quantitative data about infectivity, pathReceived for publication 3 February 1970. t The Rockefeller University, New York, New York 10021. +t Please address reprint requests to author Dickson D. Despommier. * This work was supported, in part, by USPHS Training Grant F02-A-142057, USPHS Grant AI-04842, and NIH Post-Doctoral Fellowship 1F2-AI--31, 188. ogenicity, and immunogenicity of the newborn larva of T. spiralis are still lacking. We describe here a reproducible system for obtaining newborn larvae of a known age in large numbers. Isolation techniques are described, and a quantitative comparison is made of the infectivity of pure preparations of larvae inoculated into rats via the intraperitoneal and intravenous routes. We show that both mice and rats can be infected with this stage via the ip and iv routes and that the newborn larvae are more infective for the rat when they are inoculated iv as opposed to ip. MATERIALS AND METHODS Mature muscle larvae were obtained from stockinfected CFW male mice by the method of Larsh and Kent (1949). Male Wistar rats (120 g) were inoculated orally with approximately 10,000 muscle larvae suspended in a mixture of 0.6% nutrient broth and 2% gelatin with the aid of a syringe fitted with a blunt 18-gauge needle. We removed the food from the rats on the 6th day postinfection and killed them on the 7th. The entire small intestine was then removed from each animal, slit longitudinally, cut into 2-cm sections, and placed in a modified Baermann apparatus containing 0.85% saline solution at 37 C. Adult worms were collected over a period of 4 hr, and resedimented in flasks, then washed 4 times at intervals of 0.5 hr with saline at 37 C. Worms thus treated appeared free of debris and were viable. The worms were then transferred to a large, glass moisture chamber containing 300 cc of the following me-

The pesticide malathion (MT), an organophosphate, is highly neurotoxic and causes cholinergic disorders as well as cytotoxicity, genotoxicity, mutagenicity, carcinogenicityand reproductive toxicity. Our purpose was to study the effect of ellagic acid (EA)and Vitamin C on the testis against MT-induced toxicity in the rats. Thirty-six adult Wistar rats were employed, separated into six groupsand were given treatment for 14 days. The toxicity of MT on the testis was evaluated using a variety of physical parameters,such as mortality rate and body weight, as well as biochemical parameters, such as total protein, total cholesterol, serum glutamic-oxaloacetic transaminaseand serum glutamic-pyruvic transaminase, and haematological parameters, such as counts of red blood cells, haemoglobin (Hb)and white blood cells, as well as mean corpuscular volume, mean corpuscular Hb, and mean corpuscular Hbconcentration. At the end of the experiment, rats were killed and a histological examination of the testis was performed. A sperm count technique and an analysis of sperm motility were used to determine the sperm quality. Biochemical indicators, sperm count, motility, viabilityand morphology were significantly decreased with MT. When compared withMT and the control group, EA and Vitamin C administration significantly increased sperm motility and count (p < 0.05). After receiving EA and Vitamin C, biochemical indicators and histological characteristics are also intensified. The results of the current investigation showthat EA and Vitamin C can both reduce increased levels of biochemical markers and improve pathological alterations in the testis brought on by MT treatment.

Lactic acid bacteria alleviate di-(2-ethylhexyl) phthalate-induced liver and testis toxicity via their bio-binding capacity, antioxidant capacity and regulation of the gut microbiota

Acetaminophen (APAP), also known as paracetamol outside the USA, is a non-steroidal anti-inflammatory drug (NSAID) and a widely used medication for over 50 years. Acetaminophen is an over-the-counter, commonly used analgesic for the relief of mild-to-moderate pain and fever, and one of the most common causes of poisoning worldwide. At therapeutic recommended dosage of up to 4 g day−1 it is safe and effective, with relatively few side-effects. However, an overdose can cause serious and even fatal liver toxicity. In the US alone, APAP accounts for up to 50% of all adult cases of acute liver failure (Amar & Schiff, 2007). In recent years, there is an on-going debate concerning whether therapeutic doses of APAP can cause acute liver toxicity and failure. A current comprehensive review based on published medical literature addressed this issue. Notably, prospective studies indicated that repeated use of a true therapeutic APAP dosage may slightly increase the levels of serum aminotransferases (markers of liver toxicity), but liver failure or death were not reported. In contrast, retrospective reports indicated heightened levels of serum aminotransferases and several studies reported an association with liver injury and death. These contradictory findings are largely attributed to inaccurate dosage information in some cases, suggesting an overdose rather then true therapeutic dosage use (Dart & Bailey, 2007). Yet, an overdose of 15 g or more in a single day can cause acute liver failure. An overdose of APAP was shown to deplete liver stores of glutathione (a major antioxidant molecule) and to promote liver injury via multiple mechanisms. In particular, depletion of glutathione can alter the hepatocyte redox balance and elicit innate immune responses. Obstructive sleep apnoea (OSA), a breathing disorder in sleep, is characterized by nightly recurrent upper airway occlusions and pauses in respiration, which result in intermittent hypoxia and sleep fragmentation. It affects more than 4–24% of males and 2–9% of females in the adult population. These values rise to 50% in the obese (Young et al. 1993). Also, OSA directly predisposes to conditions associated with chronic pain, such as headaches. Obese patients are particularly prone to muscle or joint pain. However, the most widely studied association by far is that of OSA with increased cardiovascular morbidity and mortality that is preceded by increased oxidative stress, inflammatory cell activation and atherosclerosis (Lavie, 2003). In the present issue, Savransky et al. (2009) have investigated whether chronic intermittent hypoxia (CIH) can induce liver injury in mice chronically dosed with APAP. For that purpose, the authors used a mouse model mimicking sleep apnoea in humans. In this well-designed and carefully executed study, four treatment groups of C57BL/6J mice were used, with the appropriate controls and placebo treatments [i.e. intermittent air (IA), IA + APAP, CIH and CIH + APAP]. The data presented clearly demonstrate that when combined with APAP administration, CIH greatly enhances liver toxicity, while CIH or APAP alone do not. Interestingly, the mechanisms participating in the development of liver injury in the combined treatment, i.e. oxidative stress, inflammation and cell death, were increased. Specifically, oxidative/nitrosative stress was demonstrated by a decrease in liver glutathione content and an increase in 3-nitrotyrosine, suggesting formation of toxic peroxynitrite in hepatocytes. The proinflammatory chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 were increased as well. Furthermore, liver injury, as determined by increased activity of aminotransferases and histological examination, revealed hepatocyte cell death (Fig. 1). A putative scheme describing liver injury in rodents exposed to chronic intermittent hypoxia or in patients with OSA using acetaminophen The scheme is relevant to this discussion; other pathways involving oxidative stress and inflammation which lead to atherosclerosis and cardiovascular morbidity in OSA are not shown. Since oxidative stress and inflammatory cell activation are increased in patients with OSA and the use of APAP as a pain reliever also induces oxidative/nitrosative stress and inflammation, these two converging pathways can be amplified and result in liver injury. Abbreviations: APAP, acetaminophen; CIH, chronic intermittent hypoxia; MIP-2, macrophage inflammatory protein-2; MCP-1 monocyte chemoattractant protein-1; and OSA, obstructive sleep apnoea. This is an interesting and important study with potential clinical implications for liver toxicity in patients with OSA using such common pain relievers as APAP, and possibly in other diseases associated with an intermittent hypoxia component. Given that APAP is a commonly used pain reliever, the heightened prevalence of OSA in the general population, and that OSA predisposes to several conditions associated with chronic pain, the combination of these findings suggests a potentially enhanced liver toxicity in these patients even at a recommended dosage use. This possibility seems likely because only the combined treatment of APAP with CIH lowered liver glutathione content, a finding which is in agreement with earlier findings on liver toxicity in humans due to overdose. This is further emphasized by the heightened levels of several inflammatory markers that have also been described in liver toxicity. Thus, in patients with disordered breathing or CIH, APAP use may further amplify on-going inflammatory responses (Fig. 1). That being said, the limitations of this study should be kept in mind. Firstly, the APAP dose administered was two- to fourfold higher than what is recommended as a safe dosage for humans. Could it be a case of an overdose in these mice? Moreover, various mouse strains display differential sensitivities to drugs. Specifically, the C57BL/6J strain used in this study has been shown to be more sensitive then BALB/c mice treated with the same APAP dosage. While 150–200 mg kg−1 APAP augmented liver aminotransferases and inflammatory markers in C57BL/6J mice, only a modest elevation was noted in BALB/c mice. Secondly, no less important in determining drug toxicity is the route of administration. In many tested drugs, the toxicity by intraperitoneal injection exceeds oral toxicity. In the study by Savransky and co-workers, APAP was administered by a daily 200 mg kg−1 bolus intraperitoneally. In many animal studies testing the effects of APAP, and other drugs, this is a commonly used route of administration. However, before a firmer conclusion on humans can be drawn, APAP should be administered orally (by gavage), scheduled at least twice daily, and at therapeutic doses. Yet the most important message that the Savransky et al. (2008) study brings to our attention is the possibility that OSA patients could be at a greater risk for developing liver injury by using such a common pain reliever as APAP. These animal studies should not be ignored; such data should set the stage for parallel controlled clinical trials in humans, since OSA is highly prevalent and APAP is widely used (or abused).

The effects of toxic and nontoxic compound treatments were investigated by high resolution custom developed 2-11 pH gradient NEPHGE (non equilibrium pH gradient electrophoresis) two-dimensional electrophoresis. Two models were compared: (i) in vivo rat and (ii) the human cell line HepG2, to test their suitability in a proteomics based approach to identify a toxicity marker. 163 and 321 proteins were identified from the rat liver and the HepG2 proteome. These represent various isoforms of 113 and 194 different NCBI annotated gene sequences, respectively. Nine compounds were selected to induce proteome variations associated with liver toxicity and metabolism. The rat liver proteome database consists of 78 gels, the HepG2 database of 52 gels. Variant proteins were assessed regarding their usefulness as a toxicity marker by evaluating their treatment specificity against multiple control treatments. Thirteen potential toxicity marker proteins were found in rat liver and eight in HepG2. Catalase and carbamoylphosphate synthetase-1 isoforms were found to be significantly changed after treatment by 4/4 and 3/4 toxic compounds in rat liver, respectively. Aldo-keto-reductase family 1, member C1 was implicated for 3/4 liver cell toxic compounds in HepG2. Our approach was able to differentiate the quality of potential toxicity markers and provided useful information for an ongoing characterization of more compounds in a wider number of toxicity classes.

Tetracarpidium conophorum (African Walnut) is a plant with acclaimed multi-therapeutic properties in different parts of the plant. This research investigated the effect of fermented walnut supplemented diet on cadmium-induced toxicity in the liver and brain of rats. Twenty male Wistar rats were divided into four groups of five animals each weighing between 90-140 g. Group 1 received 5 mg/kg body weight cadmium chloride (CdCl2) and normal rat feed. Group 2 received a normal rat diet while groups 3 and 4 received 5 mg/kg body weight of cadmium chloride, and 5% and 10% walnut supplemented feed respectively. Cadmium (Cd) was administered daily for 6 weeks by oral intubation. Rats were sacrificed 24 hrs after the final treatment. Cd exposure elicited increased activities of Acetylcholinesterase, Superoxide dismutase, Catalase as well as elevated Glutathione levels. In addition, Cd exposure caused increases in rat plasma cholesterol and triglyceride concentration. The fermented walnut supplemented diet restored some rats’ biochemical parameter to near normal comparable to control. Our study shows that walnut supplemented food could substantially moderate Cd-induced toxicity in rat liver and brain while providing health and nutritional benefits. Hence, it could be useful for occupationally exposed individuals as a dietary intervention to reduce adverse health effects.

Protocatechuic acid (PCA) possesses numerous pharmacological activities, including antioxidative and anti-inflammatory activities. This study seeks to investigate its underlying mechanism of action in the liver and brain toxicity induced by CCl4 in male albino rats. Rats were given PCA at 10 and 20mg/kg daily and orally as a pretreatment for seven days. A single injection of CCl4 was given 2h later to induce brain and liver toxicity. CCl4 moderately elevated the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). PCA lowered AST level significantly when compared to control. Total protein and albumin levels presented insignificant changes (p>0.05) in all groups while lipid profile showed increased total cholesterol level and reduced high-density lipoprotein (HDL) by CCl4. PCA (10mg/kg) significantly reduced the cholesterol level while the 20mg/kg dose moderately prevented HDL reduction. There was an increased MDA production with a corresponding low GSH level in the group treated with CCl4. Activities of superoxide dismutase, catalase, and glutathione-S-transferase in both organs also declined. PCA, especially at 10mg/kg attenuated lipid peroxidation by increasing GSH level in the organs. Biochemical assays revealed the improvement of antioxidant enzyme activities by PCA in these organs. Furthermore, PCA lowered the level of proinflammatory cytokine COX 2 in the brain and liver while NF-kB expression was inhibited in the brain. Histopathology reports validated the effects of PCA. PCA exhibited protection against toxicity in these tissues through antioxidant and anti-inflammatory activities and the potential mechanism might be through modulation of the NF-κB/COX-2 pathway.

We have previously addressed the question of whether the attenuating mutations of domain V of the Poliovirus IRES were specific for a given genomic context or whether they could be extrapolated to a genomic related virus, the Coxsackievirus B3 (CVB3). Accordingly, we have described that Sabin3-like mutation (U473→C) introduced in the CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we assessed the protection provided by the Sabin3-like mutant against CVB3 infection. For this purpose, we analyzed, in vivo, the Sabin3-like phenotype in Swiss mice inoculated with CVB3 and CVB4 E2 prototype strains either by oral or intraperitoneal (i.p) routes and explored the capacity of this mutant to act as a vaccine vector after the challenge. The Sabin3-like RNA was detected by semi-nested PCR in different organs: heart, pancreas and intestine at 10 days post-inoculation with both oral and i.p routes. Additionally, we did not observe any histological alterations in heart and intestine tissues. RNA was detected in the different organs of all mice immunized with the Sabin3-like strain and challenged with either CVB3 or CVB4 E2 by oral route at 7 days post-challenge. In contrast, no histological alteration of heart or pancreas tissues was observed after challenge with both wild-strains. Interestingly, the detection of viral RNA in heart, pancreas and intestine of mice immunized by i.p route was negative at 7 days post-challenge with CVB3 and CVB4 E2, and mice were protected from myocarditis and pancreatitis.

Zearalenone (ZEA), an estrogenic mycotoxin, is produced mainly by Fusarium fungi. Previous studies indicated that acute ZEA exposure induced oxidative stress and damage in multiple organs. Therefore, the present study was designed to investigate the adverse effects of dietary ZEA (1.1 to 3.2 mg/kg of diet) on oxidative stress and organ damage in postweaning gilts. A total of 20 gilts (Landrace × Yorkshire × Duroc) weaned at d 21 with an average BW of 10.36 ± 1.21 kg was used in the study. Gilts were housed in a temperature-controlled room, divided into 4 treatments, and fed a basal diet only (control) or basal diet supplemented with purified ZEA at a dietary concentration of 1 (ZEA1), 2 (ZEA2), or 3 (ZEA3) mg/kg of diet for 18 d ad libitum. The actual ZEA contents (analyzed) were 0, 1.1 ± 0.02, 2.0 ± 0.01, and 3.2 ± 0.02 mg/kg for control, ZEA1, ZEA2, and ZEA3, respectively. Gilts fed different amounts of dietary ZEA grew similarly with no difference (P > 0.05) in feed intake. Vulva size increased linearly over the 18 d of feeding in gilts fed diets containing 1.1 mg of ZEA/kg or greater (P < 0.001). Relative weight of genital organs, liver, and kidney increased linearly (P < 0.05) in a ZEA-dose-dependent manner. Serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamate transferase, urea, and creatinine (P < 0.05), and malondialdehyde concentrations in both serum and liver (P < 0.001) were also increased linearly in a ZEA-dose-dependent manner. However, spleen relative weight (P = 0.002) and activities of total superoxide dismutase and glutathione peroxidase (in both serum and liver (P < 0.05) were decreased linearly as dietary ZEA increased. Results showed that besides genital organs, the liver, kidney, and spleen may also be target tissues in young gilts fed diets containing 1.1 to 3.2 mg of ZEA/kg for 18 d. Increased key liver enzymes in the serum suggest progressive liver damage caused by feeding ZEA, and an increase in oxidative stress in gilts is another potential impact of ZEA toxicity in pigs.

Drug reaction with eosinophilia and systemic symptoms. is a very dangerous adverse drug effect causing rashes, eosinophilia, and multiple organ damage. Many drugs are implicated in causing DRESS with most common ones being antimicrobials and antiepileptics. Dapsone used in the treatment of Hansen’s disease as a first-line agent is known for causing many side effects ranging from nausea, vomiting, insomnia, anaphylaxis, hypersensitivity reactions, rashes, muscle weakness, abdominal pain, and so on. Hence, we report a rare case of dapsone-induced DRESS in a tertiary care hospital in South India.Keywords: Dapsone, Adverse effect, Liver toxicity, Rashes, Eosinophilia.

We studied 160 Hepatitis C virus (HCV)-positive patients with NHL (59 indolent NHL, 101 aggressive). Median age was 67 years. HCV-RNA was present in 146. HBsAg was positive in seven patients. At diagnosis, ALT value was above UNL in 67 patients. One hundred and twenty patients received an anthracycline-based therapy, alkylators, 28 received chemotherapy plus rituximab. Cytotoxic drugs dose was reduced in 63 patients. Among 93 patients with normal ALT at presentation, 16 patients developed WHO grade II-III liver toxicity. Among 67 patients with abnormal ALT, eight patients had a 3.5 times elevation during treatment. Among 28 patients treated with rituximab and chemotherapy, five patients (18%) developed liver toxicity. Thirty four patients (21%) did not complete treatment (eight for liver toxicity). Median progression-free survival (PFS) for patients who experienced liver toxicity is significantly shorter than median PFS of patients without toxicity (respectively, 2 years and 3.7 years, P = 0.03). After a median F-UP of 2 years, 32 patients died (three for hepatic failure). A significant proportion of patients with HCV+ NHL develop liver toxicity often leading to interruption of treatment. This could be a limit to the application of immunochemotherapy programs. HCV+ lymphomas represent a distinct clinical subset of NHL that deserves specific clinical approach to limit liver toxicity and ameliorate survival.

Background and Objective: Cyclophosphamide (CYP), a commonly used chemotherapeutic agent, is administered to patients to treat various cancer types. The toxic effects of this substance are widely known and can cause damage to the liver, kidneys, nervous system and bone marrow. Metformin (MET) is frequently prescribed medication for diabetes mellitus type-2, known for its effectiveness in managing blood sugar levels. Present research aims to study the effect of co-administering MET on CYP-induced hypothyroidism by determining the level of thyroid hormones. Materials and Methods: Forty rats, with a body weight ranging from 220 to 250 g, were divided into four groups of ten animals, each for control and treatment purposes. Saline was given to animals in control group. The CYP-treated group was administered four doses of CYP (100 mg/kg each) by intraperitoneal route. The MET administered animals received 3 mg/mL dose mixed in their drinking water, starting one day preceding to the CYP jab and continued until the last dose. Further, the combination group of animals was administered four doses of a CYP and a daily MET through their drinking water. Daily observations were made on the animals for any signs of mortality, while blood samples were collected to measure TSH as well as thyroid hormones (free and total T3 and T4 concentrations). Results: The study’s data suggested that the administration of CYP resulted in a higher mortality rate than the control animals. Additionally, when combined with MET, CYP further decreased the survival rate. Furthermore, a significant reduction in the concentrations of free and total T3 and T4 were observed in animals treated with CYP and CYP+MET upon comparison with control group. On the other hand, no significant changes in TSH level were recorded across all groups. Conclusion: The findings of the study revealed that the induction of hypothyroidism by CYP was not reduced by co-treatment with MET. This suggests that while MET does have anti-proliferative properties but the toxic effects of CYP was augmented, when used together.