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

Asim Amitabh Sahu; Ankita Mukherjee; Satendra Kumar Nirala; Monika Bhadauria

doi:10.1093/toxres/tfae201

. 2024 Dec 17;13(6):tfae201. doi: 10.1093/toxres/tfae201

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

Asim Amitabh Sahu ¹, Ankita Mukherjee ², Satendra Kumar Nirala ³, Monika Bhadauria ^4,^✉

PMCID: PMC11650506 PMID: 39698395

Abstract

Background: Cyclophosphamide, an immunosuppressive alkylating agent, has been used against breast cancer, lymphoma and myeloid leukemia. Despite various therapeutic uses, its toxic impacts on multiple organs remains to be fully elucidated. Aim: This study aimed to investigate dose dependent toxic impact of cyclophosphamide on liver, kidney, brain and testis emphasizing serum and tissue biochemical and histological alterations. Materials and methods: Experimental design consisted of five groups of albino rats. Group 1–5 were administered vehicle for five consecutive days. On 6^th day, group 1 received vehicle only and termed as control; group 2–5 received cyclophosphamide through intraperitoneal route at the rate of 50, 100, 150 and 200 mg/kg dose, respectively. After 24 h of the last administration, rats were euthanised; serum and tissue biochemistry; histology, sperm count and its motility were performed. Results: Serological, biochemical and histological indices exhibited dose dependent deviations from their regular status as a marker of toxicity in liver, kidney, brain and testis. Tukey’s HSD post hoc test revealed maximum damage in multiple organs with 200 mg/kg dose of cyclophosphamide.

Keywords: Cyclophosphamide, Oxidative stress, Alkylating agent

Introduction

Chemotherapeutic approaches significantly raised survival rates against several cancer types; however, anticancer medications have a dose–response impact. Alkylating agents,¹ platinum analogues,² antimetabolites,³ topoisomerase interaction agents,⁴ anti-microtubule agents⁵ and antibiotics⁶ are such chemicals, which have recently been employed in anticancer treatment; however, these have low pharmaceutical indices. As a result, multiple exposure of higher doses is needed to exert anticancer effect with substantial toxicity that impose great agony to patients. Destroying tumour cells without affecting normal cells depends on the drug's selectivity; thus, non-specificity of anticancer agents causes severe systemic toxicity that culminates into worse condition after continuous treatment.⁷

Liver injury is a product of side effects of many anticancer drugs as they impose considerable stress leading to liver damage or even liver failure.⁸ Certain anticancer drugs lead acute kidney injury (AKI) or chronic kidney disease (CKD).⁹ Direct cellular damage, formation of toxic metabolites, reduced blood flow and immune mediated reactions are some examples by which anticancer drugs exert their toxic effects on kidney.¹⁰ Some anticancer drugs cause brain toxicity, which is known as chemotherapy-induced cognitive impairment or ``chemo brain.'' This condition refers to a range of cognitive impairments that cancer patients experience during or after chemotherapy. Reasons for chemo brain are inflammation, neurotransmitter disruption and oxidative stress.¹¹ Menstrual irregularities, premature menopause in female and oligospermia and infertility in male are also major side effects of anticancer drugs.¹² Alkylating anticancer drugs are one of the major drugs, responsible for gonadal dysfunction and mutagenic alterations in germinal cells.¹³

Cyclophosphamide, an oxazaphosphorine alkylating agent, is commonly used to treat a variety of malignancies, including solid tumour, malignant lymphoma, multiple myeloma and breast cancer.¹⁴ Approximately 80% of cyclophosphamide is biotransformed into acrolein and phosphoramide mustard¹⁵ by cytochrome P450 system in liver.¹⁶ About 32% of cyclophosphamide remains unchanged, while 68% is eliminated through kidney as cyclophosphamide metabolites within 48 hours of its administration.¹⁶^,¹⁷ Acrolein generates free radicals that impair cellular defence system to counteract reactive species,¹⁸^,¹⁹ whereas phosphoramide mustard causes damage to DNA by forming adducts with guanine.²⁰ Despite its anticancer properties, it imposes damage to liver, kidney, brain, heart, and reproductive organs.²¹^,²² It damages immune system and exerts immunosuppressive effects by inhibiting cell proliferation, non-specifically killing small lymphocytes, impairing macrophage response. The potential of cyclophosphamide for organ toxicity depends on its usage, dosage and duration of treatment.²³ This investigation focussed on the evaluation of cyclophosphamide-induced dysfunctions in liver, kidney, brain, and testis based on its dose dependent acute toxic impact on biochemistry and histology in rat model.

Materials and methods

Experimental animals and chemicals

Wistar strain of adult male rats of 150 ± 10 g body weight were procured from a laboratory animal supplier M/S Chakraborty Enterprise, Kolkata (CPCSEA Reg No: 1443/PO/Br/11/CPCSEA) and housed in the institutional animal house. Experiments was duly approved by Institutional Animal Ethics Committee (994/Ere/Go/06/CPCSEA) of Guru Ghasidas Vishwavidyalaya, Bilaspur, India. Animals were housed in polypropylene cages with bedding of rice husk, provided pelleted feed, and water made available ad libitum and maintained under standard husbandry conditions of 25 °C (± 2 °C) temperature, 12 h light and dark cycle each, and 70% ± 5% relative humidity. Cyclophosphamide (CAS number: 6055-19-2; purity: > 98.0%) was procured from TCI chemicals, Hyderabad, India. Other chemicals were of pure and analytical grade and procured from Sigma-Aldrich Company, New Delhi, Hi-media Laboratories Ltd, Mumbai and SRL chemical, Mumbai, India. Commercial diagnostic kits used in the serological analysis, including aspartate aminotransferase (Ref no: IN01–276, Lot no: I01B015), alanine aminotransferase (Ref no: IN01–278, Lot no: I01B009), cholesterol (Ref no: IN01–179, Lot no: I01B017) and triglyceride (Ref no: IN01–367, Lot no: I01B002) were procured from INDO-MEDX, Bangalore, Karnataka, India; γ-glutamyl transferase (Ref no: 1001185, Lot no: GT04301) and lactate dehydrogenase (Ref no: 9001609; Lot no: LD04358) were procured from Peerless Biotech Pvt. Ltd, Chennai, Tamil Nadu, India.; urea (Ref no: 81LS200–66, Lot no: 4000015427) and uric acid (Ref no: 82LS200–50, Lot no: 4000015941) were procured from Arkray, Mumbai, Maharashtra, India; and creatinine (Ref no: 120246, Lot no: B091303) was procured from Erba Mannheim, Mumbai, Maharashtra, India.

Experimental design

Thirty rats were divided into five groups containing six in every group. Group 1 to 5 were administered normal saline as vehicle from day 1 to 5 and group 1 served as control. On day 6, all the animals of groups 2–5 received 50, 100, 150 and 200 mg/kg dose of cyclophosphamide intraperitoneally, respectively as per previous reports.¹⁴^,^24–26 Intraperitoneal route of exposure is the most preferred route in toxicological study for many reasons as it allows faster absorption of drugs than subcutaneous, intramuscular or oral routes, bypass first pass metabolism by gut, easy to administer drugs to small animals, can handle large volumes and many more.^27–29 On day 7, all the animals were euthanised, blood was collected, serum was isolated, liver, kidney, brain, and testis were excised and small pieces of <1 cm³ were fixed in Bouin’s fixative for histology; simultaneously, samples from all organs were stored at −20 °C for biochemical analysis.

Serology

Blood was collected in glass vials and serum was separated by centrifugation at 500xg centrifugal force applied for 15 mins. Markers of liver functions, including aspartate amino transferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (γ-GT), lactate dehydrogenase (LDH), triglyceride and cholesterol and kidney functions, including urea, uric acid, creatinine were performed on semi-automatic biochemistry analyser (Meril India Pvt. Ltd) as per directions given on the manual of diagnostic kits.

Sperm count and motility test

Briefly, after removal of epididymis from testis, cauda part was separated and rinsed with phosphate buffer saline for collection of sperm suspension. The sperm suspension was used for counting sperm with the help of Neubauer haemocytometer.³⁰ Sperm motility was assessed as per WHO guidelines.³¹^,³² Around 200 sperms were observed randomly under light microscope; number of motile sperms were calculated and expressed in percentage.

Preparation of hepatic microsomes

Microsomes were prepared by calcium precipitation method.³³ Liver tissues, perfused with saline, were homogenised with 10 mM tris–HCl buffer. Prepared microsomes were stored at −70 °C for assessment of CYP2E1 activity and microsomal lipid peroxidation.

Assessment of microsomal CYP2E1 activity

The CYP2E1 activity was evaluated in terms of aniline hydroxylase (AH) activity.³⁴ About 1 mL rection mixture consisting of 0.2 M tris-acetate buffer, 25 mM MgCl₂, 1.2 mM NADPH, and 80 mM aniline was mixed with 200 μL microsomal fraction and incubated at 37 °C for 20 mins. To this rection mixture, 30% TCA was added and centrifuged at 500xg for 10 min at room temperature. Collected supernatant was then mixed with 30% Na₂CO₃ and 2% phenol, and incubated in dark for 30 min. Optical density of blue-coloured compound so formed was recorded at λ 630 nm against blank.

Assessment of oxidative stress

Lipid peroxidation (LPO) is a biomarker for assessing oxidative stress. Liver, kidney, brain, and testis tissues were homogenised in KCl solution (0.15 M) and thiobarbituric acid was used as a colouring reagent. Thiobarbaturic acid reactive substances (TBARS) so formed were calculated spectrophotometrically at λ 535 nm.³⁵ For assessment of reduced glutathione (GSH), tissue samples were homogenised in sucrose solution (1%) and a coloured compound was formed after the reaction of Ellman’s reagent. Optical density of this coloured compound was recorded at λ 412 nm.³⁶

Assessment of antioxidant enzymes

Tissue homogenates were prepared in normal saline to determine the activity of superoxide dismutase (SOD) and catalase. The SOD activity was recorded at λ 480 nm³⁷ and catalase activity was recorded at λ 240 nm.³⁸ Tissues were homogenised in 1.15% KCl for determination of GSH cycle enzymes, namely glutathione-S-transferase (GST),³⁹ glutathione reductase (GR),⁴⁰ glutathione peroxidases (GPx)⁴¹ and glucose-6-phosphte dehydrogenase (G6PDH).⁴² The level of GST, GR, GPx and G6PDH were analysed by recording optical density at λ 340 nm.

Assessment of total protein contents

Total tissue proteins of liver, kidney, brain, testis, and microsomes were calculated by method of Lowry et al. All the tissue homogenates were prepared in hypotonic solution and Folin–Ciocalteu reagent was used to develop blue-coloured compound then optical density was recorded at λ 625 nm.⁴³

Assessment of cholesterol level

Tissues were homogenised in hypotonic solution to determine cholesterol level. The FeCl₃ solution (0.1%) was used to develop coloured compound then optical density was recorded at λ 560 nm.⁴⁴

Histological preparations

Fixed tissues were processed to prepare paraffin blocks and 5 μm thickened sections were cut with rotary microtome. Haematoxylin-eosin (H-E) stained slides were observed under compound light microscope with cimos camera attachment.⁴⁵

Statistical analysis

The results were subjected to one way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc analysis considering significance at P ≤ 0.05. Data are expressed as mean ± standard error. Statistical analysis was performed with the help of SPSS statistical software package (SPSS Inc, Chicago, IL).

Results

Serology

Table 1 shows alterations occurred in serological endpoints after exposure to cyclophosphamide. Concentration of AST was significantly increased in dose dependent manner in all cyclophosphamide exposed groups as compared to the control group. Group 4 and 5 showed significant difference in AST level from rest of the cyclophosphamide treated groups as analysed by Tukey’s post hoc test. Significant enhancement in concentration of ALT and γ-GT in group 2 indicated cyclophosphamide induced toxicity. Tukey’s post hoc test revealed that 200 mg/kg dose of cyclophosphamide increased concentration of these two enzymes significantly to a maximum extent in comparison to rest of the doses. Concentration of LDH and cholesterol were significantly raised after acute exposure to cyclophosphamide at 150 and 200 mg/kg doses as compared to control. Concentration of triglyceride in serum was increased significantly after 100, 150 and 200 mg/kg doses of cyclophosphamide. Tukey’s post hoc test revealed that 200 mg/kg dose exerted significant and maximum damage to liver among four doses of cyclophosphamide.

Table 1.

Effect of different doses of cyclophosphamide on markers of liver and kidney function test.

Parameters	Control	CP 50	CP 100	CP 150	CP 200	ANOVA
AST (IU/L)	42.3 ± 2.33	82.6 ± 4.57^a	155 ± 8.57^a^,^b	211 ± 11.7^a^–^c	239 ± 13.3^a^–^c	101^d
ALT (IU/L)	29.2 ± 1.61	35.7 ± 1.97	47.7 ± 2.63^a^,^b	59.5 ± 3.29^a^–^c	85.6 ± 4.73^a^–^c^,^e	63.9^d
GGT (IU/L)	19.9 ± 1.10	23.7 ± 1.31	31.1 ± 1.72^a^,^b	32.7 ± 1.81^a^,^b	48.9 ± 2.70^a^–^c^,^e	45.5^d
LDH (IU/L)	23.7 ± 1.31	25.3 ± 1.40	30.2 ± 1.67	58.1 ± 3.21^a^–^c	65.6 ± 3.63^a^–^c	78.2^d
CHOL (mg/ dl)	27.1 ± 3.50	35.3 ± 3.91	38.6 ± 4.34	43.6 ± 4.92^a	70.2 ± 6.97^a^–^c^,^e	30.3^d
TG (mg/ dl)	63.4 ± 1.50	70.8 ± 1.95	78.6 ± 2.13^a	89.1 ± 2.41^a	126 ± 3.88^a^–^c^,^e	51.1^d
Urea (mg/ dl)	20.6 ± 1.13	29.7 ± 1.64^a	32.4 ± 1.79^a	51.7 ± 2.86^a^–^c	59.3 ± 3.28^a^–^c	59.8^d
Uric acid (mg/ dl)	1.65 ± 0.09	1.73 ± 0.10	2.19 ± 0.12^a	2.71 ± 0.15^a^–^c	3.17 ± 0.17^a^–^c	29.6^d
Creatinine (mg/ dl)	0.61 ± 0.03	0.65 ± 0.03	0.68 ± 0.04	0.72 ± 0.04	1.21 ± 0.07^a^–^c^,^e	36.9^d

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Data are presented as mean ± SE (n = 6);

Abbreviation: CP 50: Cyclophosphamide at 50 mg/kg; CP 100: Cyclophosphamide at 100 mg/kg; CP 150: Cyclophosphamide at 150 mg/kg and CP 200: Cyclophosphamide at 200 mg/kg.

Control versus CP 50

CP 50 versus CP 100

CP 100 versus CP 150

Represents significant value of ANOVA at P ≤ 0.05.

CP 150 versus CP 200 P ≤ 0.05

Concentration of urea was significantly enhanced after exposure to all doses of cyclophosphamide as compared to control group. Except 50 mg/kg dose of cyclophosphamide, all of the doses increased concentration of uric acid significantly. Although there was no significant difference between 150 and 200 mg/kg dose groups, maximum increase in concentration of both urea and uric acid was noticed with 200 mg/kg dose subjected to Tukey’s post hoc test. Creatinine was found significantly raised at 200 mg/kg dose of cyclophosphamide when compared to control and other doses groups under Tukey’s post hoc test.

Sperm count and motility analysis

Significant decrease in sperm count was observed (Table 2) after acute exposure to cyclophosphamide at all doses in comparison to control. Significantly reduced sperm motility was observed after exposure to 150 and 200 mg/kg dose of cyclophosphamide. Tukey’s post hoc test revealed that 200 mg/kg was the most toxic dose, which lowered sperm count and motility.

Table 2.

Effect of different doses of cyclophosphamide on sperm count and motility test.

Groups	Sperm count (x10⁶)	sperm motility (%)
Control	5.85 ± 0.32	90.5 ± 5.00
CP 50	4.52 ± 0.25^a	84.0 ± 4.64
CP 100	3.66 ± 0.20^a^,^b	80.0 ± 4.42
CP 150	2.85 ± 0.16^a^,^b	73.5 ± 4.06^a
CP 200	1.89 ± 0.10^a^–^d	41.0 ± 2.26^a^–^d
ANOVA	57.1^e	25.6^e

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Data are presented as mean ± SE (n = 6);

Abbreviation: CP 50: Cyclophosphamide at 50 mg/kg; CP 100: Cyclophosphamide at 100 mg/kg; CP 150: Cyclophosphamide at 150 mg/kg and CP 200: Cyclophosphamide at 200 mg/kg.

Control versus CP 50

CP 50 versus CP 100

CP 100 versus CP 150

CP 150 versus CP 200 P ≤ 0.05

Represents significant value of ANOVA at P ≤ 0.05.

The CYP2E1 activity

Administration of cyclophosphamide significantly reduced AH activity (Fig. 1A). Among four doses of cyclophosphamide, 200 mg/kg dose imposed maximum toxic effect on AH activity as compared to rest of the doses as analysed by Tukey’s post hoc test.

Oxidative stress

Exposure to cyclophosphamide increased LPO in liver, kidney, brain, and testis tissues in dose dependent manner as indicated by increased TBARS level (Fig. 2A–D). Although, no significant difference was observed in liver at 50 mg/kg dose of cyclophosphamide, rest of the doses significantly increased TBARS level. All the four doses of cyclophosphamide significantly raised TBARS in kidney, brain and testis. Tukey’s post hoc test revealed that 200 mg/kg dose inflicted maximum oxidative stress to liver as compared to rest of the doses. In case of kidney, brain, and testis, 150 and 200 mg/kg doses showed similar effect as compared to 50 and 100 mg/kg doses under analysis of Tukey’s post hoc test. Tukey’s post hoc test showed that 200 mg/kg dose of cyclophosphamide significantly increased LPO as compared to 50 and 100 mg/kg doses of cyclophosphamide. No significant difference was observed between 150 and 200 mg/kg doses in all the tissues except for liver. In liver microsomes, control and 50 mg/kg dose of cyclophosphamide showed no significant difference in LPO, whereas rest of the groups exhibited increased LPO as indicated by Tukey’s post hoc test (Fig. 1B).

Fig. 2 — Lipid peroxidation of liver, kidney, brain and testes on cyclophosphamide induced damage. Data are presented as mean ± SE (n = 6); ^a control versus CP 50, ^b CP 50 versus CP 100, ^c CP 100 versus CP 150 and ^d CP 150 versus CP 200 P ≤ 0.05; ^* represents significant value of ANOVA at P ≤ 0.05. Organs liver kidney brain testis, F variance 82.9^* 55.7^* 30.9^* 29.9^*, abbreviation: CP 50: Cyclophosphamide at 50 mg/kg; CP 100: Cyclophosphamide at 100 mg/kg; CP 150: Cyclophosphamide at 150 mg/kg and CP 200: Cyclophosphamide at 200 mg/kg.

Reduced glutathione level

Dose dependent reduction in GSH occurred after acute exposure to cyclophosphamide in all four tissues (Fig. 3A–D). In liver and kidney, significant decrease in GSH was observed in all cyclophosphamide treated groups as compared to control. In case of brain, significant decrease was observed in GSH only at 150 and 200 mg/kg doses. Tukey’s post hoc test showed significant decrease in GSH in kidney and brain tissues at 150 and 200 mg/kg doses as compared to 50 mg/kg dose of cyclophosphamide. Although dose dependent decrease in GSH was observed in testis but it remained non-significant.

Antioxidant enzymes status

Enzymatic activity of SOD (Fig. 4A–D) and catalase (Fig. 5A–D) was downregulated after acute exposure to cyclophosphamide. Significant downregulation in SOD activity was observed in liver after exposure to all doses of cyclophosphamide. The 150 mg/kg dose significantly downregulated SOD as compared to 50 mg/kg dose, whereas 200 mg/kg dose significantly downregulated SOD in liver in comparison to both 50 and 100 mg/kg doses of cyclophosphamide as analysed by Tukey’s post hoc test. The 150 and 200 mg/kg dose significantly downregulated SOD activity in kidney as compared to control. Only 200 mg/kg dose of cyclophosphamide significantly downregulated SOD in kidney as compared to 50 and 100 mg/kg doses. Brain tissue showed significant downregulation in SOD activity in all the cyclophosphamide exposed groups except 50 mg/kg dose as compared to control. Tukey’s post hoc test showed significant difference between 50 mg/kg and 200 mg/kg dose of cyclophosphamide in brain tissue. In testis, all the doses except 50 mg/kg showed significant downregulation in SOD activity as compared to control group. Tukey’s post hoc test revealed significant difference among 200 mg/kg and rest of the two lower doses; however, no significant difference was observed between 150 and 200 mg/kg dose of cyclophosphamide.

Catalase activity was downregulated in liver tissue at 100, 150 and 200 mg/kg doses as compared to both control and 50 mg/kg dose. Significant downregulation was observed in kidney at 200 mg/kg dose as compared to control, 50, 100 and 150 mg/kg doses as revealed by Tukey’s post hoc test. Brain and testis tissues showed significant downregulation in catalase activity only at 100, 150 and 200 mg/kg doses in comparison to control.

GSH cycle enzymes

Dose dependent reduction was noticed in the activity of GST, GR, GPx and G6PDH of liver, kidney, brain, and testis after acute exposure to cyclophosphamide. Activity of GST, GPx and G6PDH was significantly reduced in liver after acute exposure to 150 and 200 mg/kg doses of cyclophosphamide as compared to control (Table 3). Activity of GR was significantly reduced at 100,150 and 200 mg/kg doses after acute exposure to cyclophosphamide in comparison to control. Tukey’s post hoc test revealed significant difference between 50 mg/kg and 200 mg/kg doses in all the enzyme’s level, 100 mg/kg, and 200 mg/kg in case of GST, GPx and G6PDH, and 150 and 200 mg/kg in only GST. Significant reduction in all the enzymes of GSH cycle was recorded in kidney samples (Table 4) in 150 and 200 mg/kg dose groups; however, 100 mg/kg dose was also able to reduce GR and GPx as compared to control. Although, there was significant difference between 50 and 200 mg/kg dose in reducing GR only, rest of the enzymes showed significant decrease at 200 mg/kg dose in comparison to 50 and 100 mg/kg doses. Activity of GR, GPx and G6PDH was significantly decreased in brain (Table 5) as compared to control group at all the doses of cyclophosphamide. The 200 mg/kg dose of cyclophosphamide showed significant difference from 50 mg/kg dose in case of GST, and 50 and 100 mg/kg dose in case of GR when compared with Tukey’s post hoc test. There was no significant difference among all the doses of cyclophosphamide in lowering G6PDH. Activity of GST, GPx and G6PDH was significantly reduced in testis as compared to control (Table 6). The150 and 200 mg/kg doses showed significant difference from 50 and 100 mg/kg doses but there was no significant difference between 150 and 200 mg/kg doses as per Tukey’s post hoc test.

Table 3.

Effect of different doses of cyclophosphamide on levels of GSH cycle enzymes on liver.

Groups	Liver
Groups	GST (unit/min/mg protein)	GR (μ mole NADPH oxidized/min/mg protein)	GPX (μ mole NADPH oxidized/min/mg protein)	G6PDH (μ mole product/ min/mg protein)
Control	5.53 ± 0.31	6.04 ± 0.33	5.67 ± 0.31	12.9 ± 0.71
CP 50	4.62 ± 0.25	5.75 ± 0.32	5.18 ± 0.29	12.2 ± 0.67
CP 100	3.10 ± 0.17	4.46 ± 0.25^a^,^b	4.82 ± 0.27	10.7 ± 0.59
CP 150	2.94 ± 0.16^a^–^c	3.99 ± 0.22^a^,^b	4.26 ± 0.23^a	8.64 ± 0.48^a^,^b
CP 200	2.56 ± 0.14^a^–^d	3.61 ± 0.20^a^,^b	3.33 ± 0.18^a^–^c	8.09 ± 0.45^a^–^c
ANOVA	41.1^e	19.2^e	14.3^e	15.5^e

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Data are presented as mean ± SE (n = 6)

Abbreviation: CP 50: Cyclophosphamide at 50 mg/kg; CP 100: Cyclophosphamide at 100 mg/kg; CP 150: Cyclophosphamide at 150 mg/kg and CP 200: Cyclophosphamide at 200 mg/kg; GST: Glutathione-S-transferase; GR: Glutathione reductase; GPx: Glutathione peroxidase; G6PDH: Glucose-6-phosphate dehydrogenase

Control versus CP 50

CP 50 versus CP 100

CP 100 versus CP 150

CP 150 versus CP 200 P ≤ 0.05

Represents significant value of ANOVA at P ≤ 0.05.

Table 4.

Effect of different doses of cyclophosphamide on levels of GSH cycle enzymes on kidney.

Groups	Kidney
Groups	GST (unit/min/mg protein)	GR (μ mole NADPH oxidized/min/mg protein)	GPX (μ mole NADPH oxidized/min/mg protein)	G6PDH (μ mole product/ min/mg protein)
Control	5.20 ± 0.29	7.14 ± 0.39	4.37 ± 0.24	8.48 ± 0.47
CP 50	4.81 ± 0.27	6.49 ± 0.36	4.24 ± 0.23	7.39 ± 0.41
CP 100	3.79 ± 0.21	5.70 ± 0.31^a	3.60 ± 0.20^a	7.06 ± 0.39
CP 150	3.42 ± 0.19^a^–^c	4.96 ± 0.27^a^,^b	3.14 ± 0.17^a^,^b	6.11 ± 0.34^a
CP 200	3.05 ± 0.17^a^–^c	4.53 ± 0.25^a^,^b	2.31 ± 0.13^a^–^c	5.02 ± 0.28^a^–^c
ANOVA	19.3^d	15.2^d	21.5^d	14.3^d

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Data are presented as mean ± SE (n = 6)

Control versus CP 50

CP 50 versus CP 100

CP 100 versus CP 150

Represents significant value of ANOVA at P ≤ 0.05.

Table 5.

Effect of different doses of cyclophosphamide on levels of GSH cycle enzymes on brain.

Groups	Brain
Groups	GST (unit/min/mg protein)	GR (μ mole NADPH oxidized/min/mg protein)	GPX (μ mole NADPH oxidized/min/mg protein)	G6PDH (μ mole product/ min/mg protein)
Control	4.89 ± 0.27	3.44 ± 0.19	2.12 ± 0.12	4.42 ± 0.24
CP 50	4.28 ± 0.24	2.14 ± 0.12^a	1.74 ± 0.10^a	2.77 ± 0.15^a
CP 100	3.54 ± 0.19^a	1.54 ± 0.08^a^,^b	1.47 ± 0.08^a	2.56 ± 0.14^a
CP 150	3.09 ± 0.17^a^,^b	1.28 ± 0.07^a^,^b	1.27 ± 0.07^a^,^b	2.49 ± 0.14^a
CP 200	2.81 ± 0.15^a^,^b	0.89 ± 0.05^a^–^c	1.09 ± 0.06^a^,^b	2.16 ± 0.12^a
ANOVA	19.9^d	91.7^d	25.8^d	34.7^d

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Data are presented as mean ± SE (n = 6)

Control versus CP 50

CP 50 versus CP 100

CP 100 versus CP 150

Represents significant value of ANOVA at P ≤ 0.05.

Table 6.

Effect of different doses of cyclophosphamide on levels of GSH cycle enzymes on testis.

Groups	Testis
Groups	GST (unit/min/mg protein)	GR (μ mole NADPH oxidized/min/mg protein)	GPX (μ mole NADPH oxidized/min/mg protein)	G6PDH (μ mole product/ min/mg protein)
Control	6.03 ± 0.33	2.81 ± 0.15	1.86 ± 0.10	7.10 ± 0.39
CP 50	4.91 ± 0.27^a	2.70 ± 0.15	1.07 ± 0.06^a	5.36 ± 0.30^a
CP 100	3.49 ± 0.19^a^,^b	1.59 ± 0.09^a	0.89 ± 0.05^a	4.74 ± 0.26 ^a
CP 150	2.38 ± 0.13^a^–^c	1.12 ± 0.61^a^–^c	0.58 ± 0.03^a^–^c	3.61 ± 0.20^a^–^c
CP 200	2.24 ± 0.12^a^–^c	0.97 ± 0.05^a^–^c	0.51 ± 0.03^a^–^c	3.16 ± 0.17^a^–^c
ANOVA	63.3^d	74.3^d	96.3^d	38.2^d

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Data are presented as mean ± SE (n = 6)

Control versus CP 50

CP 50 versus CP 100

CP 100 versus CP 150

Represents significant value of ANOVA at P ≤ 0.05.

Cholesterol level

Dose dependent increase in level of total cholesterol (Fig. 6A–D) and esterified cholesterol (Fig. 7A–D) was observed after acute exposure to cyclophosphamide in all the tissues. In liver, 150 and 200 mg/kg doses of cyclophosphamide not only elevated total cholesterol but also elevated esterified cholesterol as compared to control. In kidney, except 50 mg/kg dose, all higher dose groups showed significant increase in total cholesterol but 150 and 200 mg/kg dose groups showed significant increase in esterified cholesterol as comparted to control. The 200 mg/kg dose of cyclophosphamide exerted maximum damage to kidney in terms of deviation of cholesterol as compared to other cyclophosphamide treated groups under analysis of Tukey’s post hoc test. Both total and esterified cholesterol level in brain was significantly elevated at 150 and 200 mg/kg doses as compared to control. The 200 mg/kg dose of cyclophosphamide exerted maximum damage among all other doses as evident by Tukey’s post hoc test. Similarly, 150 and 200 mg/kg doses significantly increased cholesterol level as compared to 50 and 100 mg/kg doses as analysed by Tukey’s post hoc test.

Histological observations

Liver sections of control group showed regular histoarchitecture with well-arranged cord of hepatocytes, distinct nucleus, regular appearance of central vein, and sinusoidal space (Fig. 8A–C). Administration of cyclophosphamide at 50 mg/kg dose initiated liver injury in terms of dilation in sinusoidal spaces that appeared in patches around centrilobular vein and portal triads (Fig. 8D–F). The 100 mg/kg dose of cyclophosphamide was also responsible for dilation of sinusoidal space and deterioration of hepatocytes (Fig. 8G–I). Derailment in regular sinusoidal spaces and solidification of hepatocytes were clearly visible after administration of 150 mg/kg dose of cyclophosphamide (Fig. 8J–L). The 200 mg/kg dose of cyclophosphamide caused deviation of sinusoidal spaces from their regular cord like arrangements due to injury in liver sinusoidal endothelial cells and congestion of hepatocytes (Fig. 8M–O). A series of pattern of histological changes in liver were observed in a dose dependent manner after exposure to cyclophosphamide. Dilation in sinusoidal space was observed at lower doses, whereas sinusoidal obstruction syndrome was observed at 200 mg/kg of cyclophosphamide.

Fig. 8 — Histological observation of liver tissue after acute administration of cyclophosphamide. A, B and C: Control (300x, 600x and 945x respectively); D, E and F: CP 50 mg/kg (300x, 600x and 945x respectively); G, H and I: CP 100 mg/kg (300x, 600x and 945x respectively); J, K and L: CP 150 mg/kg (300x, 600x and 945x respectively); M, N and O: CP 200 mg/kg (300x, 600x and 945x respectively). Abbreviation: CV: Central vein; H: Hepatocytes; SS: Sinusoidal space; PT: Portal triad; NU: Nucleus of hepatocyte.

Kidney sections of control group showed regular histoarchitecture of glomerulus, Bowman’s capsule and renal tubules (Fig. 9A–C). After 50 mg/kg dose of cyclophosphamide, dilation in lumen of tubules, enlargement in glomerulus, and reduction in glomerular space were observed (Fig. 9D–F). The 100 mg/kg dose of cyclophosphamide showed extensive tubular atrophy, vacuolation in cytoplasm with hyalinisation (Fig. 9G–I). At 150 mg/kg dose, severe degeneration in capillary endothelial cells of glomerular capillary tufts and hyalinisation were observed, vacuolation, widening of space around collecting duct was observed as necrotic features (Fig. 9J–L). The 200 mg/kg dose of cyclophosphamide severely affected histoarchitecture of kidney in terms of degeneration of capillary endothelial cells of glomerular capillary tufts and luminal atrophy. Bowman’s capsules were severely damaged forming wide glomerular space due to overwhelming disintegration in glomerulus and lumen (Fig. 9M–O).

Histoarchitecture of brain of control group exhibited prominent and characteristic nuclei of cell body of neurons (Fig. 10A–C). Administration of 50 mg/kg dose of cyclophosphamide resulted spongiform changes in cerebral region of brain with appearance of pyknotic nuclei (Fig. 10D–F). At 100 mg/kg dose, size of spongiform changes and number of pyknotic nuclei gradually increases with appearance of dead neurons (Fig. 10G–I). The 150 mg/kg dose responsible for significant spongiform changes, vacuolation and neuronal loss (Fig. 10J–L). At 200 mg/kg dose, neuronal damage with pyknotic nuclei, and damage to endothelial lining of blood vessel were clearly observed (Fig. 10M–O).

Histological features of testis of control group showed regular histoarchitecture with normal seminiferous tubule, spermatocytes, spermatids, and appearance of thread-like structure of sperms (Fig. 11A–C). Administration of cyclophosphamide at 50 mg/kg dose slightly decreased sperm concentration in lumen of seminiferous tubule (Fig. 11D–F). The 100 and 150 mg/kg doses of cyclophosphamide gradually decreased sperm concentration, induced atrophy to germinal epithelium or different spermatocytes present in seminiferous tubules (Fig. 11G–I for 100 mg/kg, Fig. 11J–L for 150 mg/kg groups). At 200 mg/kg doses of cyclophosphamide, prominent damage to basement membrane of seminiferous tubules, intense damage in spermatocytes and severe reduction in tuft of sperm were observed (Fig. 11M–O).

Fig. 11 — Histological observation of testis tissue after acute administration of cyclophosphamide. A, B and C: Control (300x, 600x and 945x respectively); D, E and F: CP 50 mg/kg (300x, 600x and 945x respectively); G, H and I: CP 100 mg/kg (300x, 600x and 945x respectively); J, K and L: CP 150 mg/kg (300x, 600x and 945x respectively); M, N and O: CP 200 mg/kg (300x, 600x and 945x respectively). Abbreviation: ST: Seminiferous tubule; SL: Lumen of seminiferous tubules; SC; spermatocytes; S: Sperms; notched arrow head: Damage to plasma membrane.

Discussion

Liver is the major site of biotransformation of xenobiotics that occasionally produces hazardous metabolites.⁴⁶ Kidney is a susceptible organ and prone to damage as many drugs are excreted out through it.⁴⁷ Certain drugs or their metabolites cross blood–brain barrier and blood-testes barrier and exert toxic effects. One of such drugs is cyclophosphamide, which is hazardous to multiple organs.⁴⁸ Cyclophosphamide induced toxicity has been reported at higher doses, which causes severe damage to kidney, gonads and bone marrow.^49–52 However, lower doses are associated with reversible tissue damage mainly to liver.²⁴ There is scarce understanding on toxic impact of cyclophosphamide at lower to higher dose range on multiple organs under acute exposure. Thus, in the present study, we examined toxic impact of cyclophosphamide on liver, kidney, brain, and testis at a series of doses by assessing biochemical changes in serum, liver, kidney, brain and testis along with histology of these four organs.

Exposure to cyclophosphamide resulted increase in concentration of AST, ALT, γ-GT in serum that could be due to alteration in transport mechanism, breakdown of amino acids and alteration in permeability of cell membrane, which allowed leakage of these enzymes into circulation.⁵³ One of the key roles of γ-GT is to induce Sertoli cells of testis for maturation and replication.⁵⁴ Impairment in this enzyme could lead disruption in spermatogenesis, which was supported by histological observations of testis and sperm count. Elevated level of serum and tissue cholesterol and serum triglycerides revealed alterations in lipid metabolism and plasma membrane as a toxic effect of cyclophosphamide. Increase in urea, uric acid and creatinine suggested impairment in kidneys due to cyclophosphamide induced toxicity, which are consistent with earlier reports.^55–57 Damage in glomerulus, as observed in histology, impaired glomerular filtration that resulted release of these markers into blood circulation after exposure to cyclophosphamide. It may be postulated that cyclophosphamide increased metabolism of muscle tissue, which increased level of creatinine.⁴⁸ The LDH is a cytoplasmic enzyme, which is found in almost all tissues, including muscle, liver, and kidney. Elevation in serum LDH proposed alteration in cellular permeability due to exposure to cyclophosphamide.⁵⁸

Sperms are highly susceptible to damage by ROS.⁵⁹ High concentration of PUFA and low availability of antioxidants could be the reason of decrease in sperm count, motility and increase in the morphological defects with consecutive increase in doses of cyclophosphamide. Previous reports corroborate our findings suggesting reduction in sperm count due to drug induced acute toxicity.^60–62 Damage due to ROS generation is also evident by damage in seminiferous tubules, detachment of spermatogonia, spermatocytes, spermatids, and destruction of Leydig cells.

Member of heme protein superfamily, the cytochrome P450, catalyses metabolism of numerous endogenous and exogenous substances to transform them into more polar metabolites.⁶³ In our study, AH activity was evaluated to assess expression of CYP2E1, as its activity was primarily mediated by CYP2E1. Acrolein, the metabolite of cyclophosphamide, induces stress in endoplasmic reticulum that hampered protein synthesis, which might be the reason behind decrease in CYP2E1 levels and corresponding AH activity with respect to increase in doses.⁶⁴^,⁶⁵

Overwhelming production of ROS after exposure to cyclophosphamide resulted imbalance between ROS and antioxidant defence machinery. Formation of peroxide radicals increased lipid peroxidation in all the tissues.⁶⁶ The GSH plays a major role in protecting cells against ROS generated cell injury. The metabolites of cyclophosphamide, mainly acrolein and 4-hydroxycyclophosphamide form conjugate with the GSH; thus, lowered GSH reserves.⁵⁴ Depletion in GSH lowered cellular defence of tissues against free radicals and enabled them susceptible to ROS.⁶⁷ The SOD scavenges superoxide radical and form reactive nonradical hydrogen peroxide, which is detoxified by catalase and GPx to form non-reactive water and molecular oxygen.⁶⁸ The enzymes involve in GSH metabolism (GPx, GR, G6PDH, GST) also help in protection of cell from ROS generated cell injury. The GPx catalyses reaction of two molecules of GSH to GSSG by donating two hydrogen ions. The GR is responsible for regeneration of GSH from GSSG with concomitant release of NADP⁺; thus, restored its level. The GST conjugate with cyclophosphamide reactive semiquinone metabolite to induce their detoxification, metabolism and elimination.⁶⁹ In our study, marked decreased in these enzymes suggested elevated oxidative stress after cyclophosphamide exposure. This decrease in the enzymes level of GPx, GR, G6PDH and GST might be due to formation of acrolein-GSH adduct that further initiated a cascade reaction or affected antioxidant enzymes after exposure to cyclophosphamide.

Various biochemical variables of serum, liver, kidney, brain and testis and histological observations of these four organs showed severity of damage caused by cyclophosphamide.

This onset of cyclophosphamide induced multiple organ damage could be due to generation of ROS via its metabolites: phosphoramide mustard, chloroacetaldehyde and particularly acrolein.²⁵^,⁷⁰^,⁷¹ Acrolein induces stress in endoplasmic reticulum that decreased CYP2E1 activity. Acroleinyl radicals, formed after metabolism of acrolein, consequently initiated lipid peroxidation.⁷²^,⁷³ The ROS generated can propagate further, initiating a chain reaction that damage cellular components such as lipid, protein and nucleic acids resulting in increase of lipid peroxidation and decrease in enzymatic and nonenzymatic antioxidants.⁷⁴ Thus, it may be proposed that acroleinyl radicals, acrolein-GSH adduct and decrease in GSH altogether are responsible for cyclophosphamide induced multiple organ injury (Fig. 12). Clinically, this cyclophosphamide induced multiple organ damage lies within alteration of serological and biochemical variables due to sinusoidal obstruction syndrome, glomerulonephritis, neuronal loss and higher vacuolation in brain, and degeneration of basement membrane of germ cell layer in testis with severe reduction in sperm count.

Fig. 12 — Proposed mechanism for cyclophosphamide induced multiple organ injury.

Our study concluded that 50 mg/kg dose of cyclophosphamide brought mild changes in organ biochemistry and histology. As liver is the main site for metabolism, it underwent damage to a higher extent as compared to rest of the tissues at the lowest dose. The 200 mg/kg dose of cyclophosphamide imposed severe injury to all the organs i.e. liver, kidney, brain and testis. Apart from cytotoxicity to cancer cell, damage of normal tissues is a regular consequence of anticancer therapy that affect normal life of a patient who receives drugs like cyclophosphamide. As cyclophosphamide is employed in the treatment of various malignancies, findings of this study may help in making strategies to form a second line of defence by use of complementary medicine in mitigating multiple organ injury. Although oxidative stress mediated toxicity remains the primary mechanism for cyclophosphamide induced toxic consequences, further study may explore more insights of mechanisms behind cyclophosphamide induced multiple organ injury.

Contributor Information

Asim Amitabh Sahu, Toxicology and Pharmacology Laboratory, Department of Zoology, Guru Ghasidas University, Ratanpur Road, Koni-Bilaspur, Chhattisgarh 495009, India.

Ankita Mukherjee, Toxicology and Pharmacology Laboratory, Department of Zoology, Guru Ghasidas University, Ratanpur Road, Koni-Bilaspur, Chhattisgarh 495009, India.

Satendra Kumar Nirala, Laboratory of Natural Products, Department of Rural Technology and Social Development, Guru Ghasidas University, Ratanpur Road, Koni-Bilaspur, Chhattisgarh 495009, India.

Monika Bhadauria, Toxicology and Pharmacology Laboratory, Department of Zoology, Guru Ghasidas University, Ratanpur Road, Koni-Bilaspur, Chhattisgarh 495009, India.

Author contributions

Asim Amitabh Sahu (manuscript writing, experimental work, revising and editing), Ankita Mukherjee (experimental work), Satendra Kumar Nirala (proof reading, manuscript finalisation, data curation, editing), Monika Bhadauria (conceptualisation of study, experimental design, data validation, editing).

Funding

None.

Conflict of interest statement. There are no conflicts of interest to declare.

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PERMALINK

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

Asim Amitabh Sahu

Ankita Mukherjee

Satendra Kumar Nirala

Monika Bhadauria

Abstract

Introduction

Materials and methods

Experimental animals and chemicals

Experimental design

Serology

Sperm count and motility test

Preparation of hepatic microsomes

Assessment of microsomal CYP2E1 activity

Assessment of oxidative stress

Assessment of antioxidant enzymes

Assessment of total protein contents

Assessment of cholesterol level

Histological preparations

Statistical analysis

Results

Serology

Table 1.

Sperm count and motility analysis

Table 2.

The CYP2E1 activity

Fig. 1.

Oxidative stress

Fig. 2.

Reduced glutathione level

Fig. 3.

Antioxidant enzymes status

Fig. 4.

Fig. 5.

GSH cycle enzymes

Table 3.

Table 4.

Table 5.

Table 6.

Cholesterol level

Fig. 6.

Fig. 7.

Histological observations

Fig. 8.

Fig. 9.

Fig. 10.

Fig. 11.

Discussion

Fig. 12.

Contributor Information

Author contributions

Funding

References

ACTIONS

PERMALINK

RESOURCES

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