Comparative Evaluation of Vehicle, Dose, and Duration-related Oxidative, Cardiotoxic, Inflammatory and Histologic Responses of Chromium 6+ and Doxorubicin in Rats’ Heart

doi:10.21203/rs.3.rs-2847266/v1

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Comparative Evaluation of Vehicle, Dose, and Duration-related Oxidative, Cardiotoxic, Inflammatory and Histologic Responses of Chromium 6⁺ and Doxorubicin in Rats’ Heart

https://doi.org/10.21203/rs.3.rs-2847266/v1

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Doxorubicin (Dox) is cardiotoxicity is established while chromium 6⁺ compound (Cr[VI]) could be cardiotoxic due to its bioaccumulation capacity. This study compared vehicle, dose, and duration-related oxidative, cardiotoxic, inflammatory and histologic responses of Cr[VI] and Dox intoxication in rats’ heart by standard protocols. The rats were respectively intoxicated with Cr [VI] and Dox in 3 different phases. In the first phase, sixty rats were assigned to six groups of ten each. Group 1 served as the Control while groups 2, 3, and 4 were treated with oral doses of 10, 20, and 30 mg/kg body weight (b.wt) of K₂Cr₂O₇ (Cr[VI]) solution while groups 5 and 6 received intraperitoneal administration of 15 and 20 mg/kg b.wt Dox for two days, respectively, before the sacrifice. The procedure was repeated in the second and third Phases, but for 60 days. Oxidative, cardiotoxic, inflammatory and histologic indices were determined in the rats’ heart. The results indicated that exposure to either Dox or Cr{VI] caused a significant (P < 0.05) dose, vehicle and duration-dependent decrease in Superoxide dismutase (SOD), Glutathione peroxidase (GPx), Catalase (CAT) activities and Nitric Oxide(NO) levels but an increase in Cardiac Troponin (CTnI) levels, Creatinine-kinase (CK-MB), C-reactive protein(CRP), Aspartate-transaminase(AST), Lactate-dehydrogenase (LDH) and Malondialdehyde (MDA) compared to the control. Heart histopathology of Dox- and Cr[VI] treated rats showed dose, vehicle and duration-dependent pulmonary oedema, hyaline necrosis and displacement of adjacent myocytes compared to control. Thus, Cr[VI] compared well with Dox in cardiotoxicity induction accompanied with oxidative stress, inflammatory and histo-hepatic responses in the rats’ heart.

Toxicology

Chromium 6+

Cardiotoxicity

Doxorubicin

Potassium dichromate

Chromium pollution is a severe threat to man and the entire ecosystem. Several researchers deduce the indiscriminate release of Cr [VI] compounds during industrial activities like photo production, chrome plating, paint making, welding, electroplating, and leather tanning as the major ways of Cr-pollution in the ecosystem (Garcia-nino et al.,2015; Onyedikachi et al.,2018; Onyedikachi et al., 2019; Hashem et al., 2020; Wuri et al., 2022; Fagundes et al. 2022. Firoozichahak et al.,2022). Cr (VI) toxicity has been implicated in various Organ toxicities like respiratory, gastrointestinal, hepatic, renal, neurological, and reproductive systems. It alters general development, causes increased ROS production which limits the activities of antioxidant enzymes, protein oxidation, and DNA damage (Bagchi et al., 2002, Awasthi et al.,2018; Husain & Mahmood, 2018; OEHHA, 2009; ATSDR, 2012; Remy & Clay, 2014; Garcia-Nino et al., 2015; Onyedikachi et al., 2019; De Almeida et al., 2021). The health effects of Cr, like other trace elements and chemicals of concern, may vary with the route of exposure, possibly, through inhalation, dermal contact, and ingestion of contaminated water and food crops grown in Cr-polluted soils (Onyedikachi et al., 2019; Das et al., 2021; Prasad et al., 2021). Chromium (Cr) has varying oxidation states and solubility, the trivalent [Cr (III)] and hexavalent [Cr (VI)] forms (Carvalho et al., 2014, Homa et al., 2016, John, B.V., 2019). However, the Cr (VI) forms are more hazardous to health because they make up very strong oxidizing agents that get absorbed easily across the membranes through non-specific anion carriers (Pokhrel, G. R.& Pokhre, G. 2022)).

Many studies have revealed that cardiotoxicity may occur due to exposure to drugs, toxic metals, and other unknown causes (Guven et al.,2018). Cardiotoxicity results in myocardial and electrophysiology dysfunctions which affects the heart ability to pump blood efficiently resulting in various co-morbidities and deaths ((Tabassum et al., 2015; Mladěnka et al.,2018; Erbaş et al.,2022). Doxorubicin, an established cardiotoxic agent, is a broad-spectrum antitumor drug used to treat various cancers but could lead to cardiac failure and cardiomyopathies even after treatment cessation ((Hardaway, 2019; Abdelatty et al.,2021; Morelli et al.,2022). The early damage occurs 1 to 3 days after drug administration, often resulting in apoptosis, necrosis, oxidative stress, which results in irreversible cardiotoxicity (Hardaway, 2019). The molecular mechanisms of DOX-induced cardiotoxicity are unclear. Although, the suggestive possible mechanisms of action of DOX include DOX-mediated attack on the mitochondria, increase in reactive oxygen species, iron ferroptosis, disruption of calcium homeostasis, topoisomerase dysregulation and elevation in cardiac enzymes AST, LDH and CK–MB, and Troponin (Fernandez-Chas et al., 2018; Al-Malky et al.,2019; Morelli et al., 2022).

Previously, high levels of Cr⁶⁺ were detected in soils and food crops grown around industrial areas in the South East of Nigeria (García-Niño et al.,2015; Onyedikachi etal., 2019). This could indicate high level of Cr[VI] contamination and possible health risks. The dearth of information on the cardio toxic effect of Cr (VI) compounds and also, the growing concerns of possible cardio-related ailments observed among the residents and industrial workers in the areas (García-Niño et al.,2015; Onyedikachi etal., 2019, OEHHA, 2009; NIOSH, 2013; Das et al.,2015; Nur-E-Alam et al.,2020) warranted the present study aimed to evaluate and compare the vehicle, dose, and duration-based oxidative, cardiotoxic, inflammatory and histologic responses of administration of Cr (VI) (as potassium dichromate) and Dox in adult male Wistar rats’ heart.

2.1 Chemicals and Drugs

Potassium dichromate (K₂CR₂O₇) was obtained from Sigma Chemical Co.(St. Loius, MO, USA). Doxorubicin and sterile water (Pfizer Drugs, USA) were purchased from Grace and Mercy Pharmacy, Umuahia, Nigeria. All other reagents were of analytical grade and were purchased from British drug houses (Poole, Dorset, UK).

2.2 Animal Husbandry

A total of one hundred and eighty adult male Wistar albino rats (age:12week and body weight:120–140 g) reared at the animal house, College of Veterinary Medicine, Michael Okpara University of Agriculture were used for this study. The animals were housed in well-ventilated plastic cages at the College of Natural Sciences, Michael Okpara University of Agriculture, Umudike, and subjected to a photoperiod of 12h light 12h dark cycle. They were allowed access to rat chow and drinking water ad libitum for 14 days. All investigations involving the experimental animals were strictly followed regarding the guidelines for the care and use of animals as defined by the National Institute of Health Committee on Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, USA (ILAR (1986).The experiment protocols were performed after the ethical approval was granted by the College of Natural Science, Research Ethics Committee (CREC/002/21), Michael Okpara University of Agriculture.

2.3. Experimental Design

Sixty (60) rats in three groups were used for each phase of treatment. The treatment phases include an acute exposure phase and two chronic exposure phases.

In the I^st phase (acute phase exposure), group 1, control rats received an equal volume of normal drinking water orally for 21 consecutive days while groups 2, 3 and 4 rats were respectively orally treated (using Gavage) with 10 mg/kg, 20 mg/kg and 30 mg/kg body weight of K₂Cr₂O₇ [Cr(VI)] compound for 21 consecutive days. Groups 5 and 6 rats respectively intraperitoneally received 15 mg/kg and 20 mg/kg body weight of doxorubicin (2 days before sacrifice).

The protocol in the 2nd phase (chronic phase exposure) was as in the 1st phase but lasted for sixty (60) consecutive days.

In the 3rd phase (chronic food supplementation phase), group 1, control rats were fed with a standard pellet diet and received an equal volume of distilled water. Group 2 rats received 0.01% of K₂Cr₂O₇ [Cr(VI)] compound in feed i.e 10mg/kg of K₂Cr₂O₇ [Cr(VI)] compound per kg body weight in 100g of Rat feed for 60 days. Group 3 rats received 0.02% of K₂Cr₂O₇ [Cr(VI)] compound in feed i.e 20mg/kg of K₂Cr₂O₇ [Cr(VI)] compound per kg body weight in 100g of Rat feed for 60 days. Group 4 rats received 0.03% of K₂Cr₂O₇ [Cr(VI)] compound in feed i.e 30mg/kg of K₂Cr₂O₇ [Cr(VI)] compound per kg body weight in 100g of Rat feed for 60 days. Group 5 and 6 rats respectively received intraperitoneal administration of 15 mg/kg and 20 mg/kg body weight of doxorubicin (2 days before sacrifice). The doses of K₂Cr₂O₇ used in this study were based on the pilot study and as previously published (Navya et al.2018; Adjroud, 2009) while doses of, and methods used for, Dox administration were based on earlier published reports (Radwan et al., 2012; Biondo, 2016; Sandamali et al.,2019; Karabulut et al., 2021 and Furihata et al.,2021).

2.4 Preparation of heart samples

All analyses, including biochemical and histopathology assessment of heart tissue carried out in the first phase, were repeated in the second and third phases. On the 22nd and 61st day, i.e., after overnight fasting. Ketamine injection was administered intraperitoneally to anaesthetize the animals, followed by prompt excision of the respective rats’ heart tissues. Part of the heart was rinsed in normal saline. One gram was weighed, sliced, and ground with laboratory mortar and pestle in a 5ml phosphate buffer solution. The resulting solution was centrifuged at 2200rpm for 25minutes in a cold centrifuge at 4⁰c. The supernatant (homogenate) was collected, labelled, and preserved in the freezer until analysis. The heart homogenates were used to assess CK-MB, CRP, AST, LDH, and Trop. Also, part of the heart was excised and fixed in 10% buffered formalin for a histopathology examination.

2.4.1 Body Weight and Relative Organ Weight

The body weight of each rat in the groups was weighed at the beginning of the study and on the day of sacrifice. The excised organs (heart) were weighed during the sacrifice to calculate the relative organ weight(using the formula below) and observed for gross lesions.

$$Relative organ weight =\frac{Organ weight \left(g\right) of animal on sacrifice day \left(g\right) }{Body weight \left(g\right) of animal on sacricice day } X 100$$

2.5 Biochemistry Assay

SOD activity was assayed by the method of Arthur and Boyne (1985). GP_X assay was carried out according to the method of Paglia and Valentine (1967). The activity of Catalase was assayed by the method of Sinha (1972). Lipid peroxidation product (MDA) was assayed according to the method described by Wallin (1993). LDH assay was carried out according to the method used by Jung (2000). CK-MB assay was carried out according to the method of Henry (1974). AST assay was according to the method of Reitman and Frankel (1975). NO assay was also carried out according to the method described by Dessy and Ferron (2004). Spectrophotometric reading was assessed using D3900 UV-VIS Spectrophotometer (Hach TechnologiesLoveland USA.)

2.6 Immuno Assays

The Troponin test (CTnI) was carried out according to the method of Melanson (2007). C-reactive proteins (CRP) were analyzed using the method of Anderson and Mccarthy (1950) as determined using commercially available ELISA kits following the procedure described in the manual.

2.7 Preparation of slides for histopathological evaluation

The hearts of the rats were isolated immediately after sacrifice, washed with cold phosphate-buffered saline (pH = 7.4) and fixed in 10% buffered formalin. Tissues were embedded in paraffin, and 5m serial sections of the heart were taken. These sections were stained with Hematoxylin and Eosin (H&E). Changes in the histopathology were observed under a light microscope (Olympus BX10, Tokyo, Japan), and images were taken using a digital camera. The individual performing the evaluation was blinded to the treatment given to the respective groups. Tissue sections of the heart were collected for histopathological studies. Prior to the commencement of tissue preparation, the heart samples were fixed in formalin (10% buffered) after washing with cold phosphate-buffered saline (7.4). The tissues were prepared by trimming and dehydrating in 70%, 80%, 90%, and absolute alcohol, clearing in 3 grades of xylene, and solidifying the tissues in molten wax. On solidifying, the tissue-containing wax blocks were cut into 5µm thick sections with a rotary microtome, floated in a water bath, and incubated at 60^˚C for 30 minutes. The 5µm thick sectioned tissues were cleared in 3 grades of xylene and rehydrated in 3 grades of alcohol (90%, 80%, and 70%). The sections were then stained with Hematoxylin for 15 minutes. Blueing was done with ammonium chloride, and differentiation was done with 1% acid alcohol before counterstaining with Eosin. Permanent mounts were made on degreased glass slides using a mountant, DPX. The prepared slides were examined with a Motic™ compound light microscope using x4, x10, and x40 objective lenses. The photomicrographs were taken using a Motic™ 5.0 megapixels microscope camera at x400 magnifications.

2.8 Statistical Analysis

The least significant difference (LSD) was used to compare differences in each sample within treatments. Data were reported as mean ± Standard Error (SE). One-way analysis of variance (ANOVA) was also used to determine significant differences between groups, considering a level of significance of less than or equal to (P < 0.05) by using SPSS. The significant difference was assessed using Duncan at (P < 0.05).

3.1 Absolute and Relative Heart Weight

The absolute and relative heart weights of rats treated orally (by administration with Gavage) with K₂Cr₂O₇ for 21 days are presented, respectively, in Appendix 1. The experimental rats' absolute and relative heart weights indicated no significant difference compared to the Control (p < 0.05). However, a slight increase in the absolute and relative heart weight of groups treated with K₂Cr₂O₇ for 60days was observed compared to the Doxorubicin and Normal (Control) groups for all treatment routes.

3.2 Different doses of K ₂ Cr ₂ O ₇ and Doxorubicin impaired Antioxidant enzymes following acute and chronic exposure to Rats in the Treatment Phases (Phase 1, 2 and 3).

Results presented in Fig. 1 (A, B and C) show that rats exposed to K₂Cr₂O₇ and Dox respectively elicited a significant (p < 0.05) decrease in GPx, CAT, and SOD activities in the treatment phases relative to the control (Group 1).

In phase 1,The SOD activities decreased by 0.89%,10.62%, 24.78% K₂Cr₂O₇ treated Rats, CAT activities also decreased by 30.86%,42.23% and 48.03% in K₂Cr₂O₇ treated rats, likewise, GPx activities decreased by 12.96%, 21.96% and 34.01% in rats treated with 10mg/kg, 20mg and 30mg/kg K₂Cr₂O₇ respectively, relative to the normal control animals. Similarly, animals treated with 15mg/kg and 20mg/kg Dox in groups 5 and 6 showed decreased GPx, CAT, and SOD activities. The percentage decreases in the enzyme activities for groups 5 and 6 relative to the control group were 2.65% and 4.24%(SOD), 0.23% and 18.33%(CAT), and 34.4% and 12.55%(GPx).

In the Second phase, the SOD activities decreased by 20%, 22.5%, 39.17% in K₂Cr₂O₇ treated rats, CAT activities also decreased by 19.34%, 30.82% and 45.62% in K₂Cr₂O₇ treated rats. Likewise, GPx activities decreased by 2.70%, 21.95% and 30.62% in Rats treated with 10mg/kg, 20mg and 30mg/kg K₂Cr₂O₇ respectively relative to the Normal control animals. Similarly, animals that received 15mg/kg and 20mg/kg Dox) in Groups 5 and 6 showed decreased GPx, CAT, and SOD activities by 10.38% and 14.17%, 1.81%, and 9.37%, 5.42%, and 8.40% respectively compared to the enzyme activities recorded for the Normal Control(Group 1).

In the Third Phase, the SOD activities decreased by 2.90%, 11.63%, and 22.67% K₂Cr₂O₇ in treated Rats. CAT activities also decreased by 4.04%, 40%, and 45% in K₂Cr₂O₇ treated rats. Likewise, GPx activities decreased by 26.92%, 35.49% and 38.11% in Rats treated with 10mg/kg, 20mg and 30mg/kg K₂Cr₂O₇ respectively relative to the normal control animals.

Similarly, animals treated with 15mg/kg and 20mg/kg Doxorubicin (Dox) in Groups 5 and 6 decreased the enzyme activities by 30.81% and 32.56% (SOD), 1.21% and 3.24%(CAT), and 27.27% and 27.47% (GPx) compared to the normal Control(Group 1).

3.3 Treating rats with different doses of K₂Cr₂O₇ and Dox induced Oxidative Stress in all the treatment phases (First, Second and Third Phases).

The results presented in Fig. 2 shows the level of MDA investigated in the hearts of the Control, K₂Cr₂O_7, and Dox treated Rats. The Rats that received both K₂Cr₂O₇ and Dox showed a significant increase in MDA levels following the First, Second and Third treatment Phases when compared to the Normal Control, the percentage increase in MDA levels for the Rats treated with 10mg/kg, 20mg/kg, and 30mg/kg K2Cr2O7 was 16.67%, respectively, 63.33%, 90.00%, respectively, in the first phase.

In the second phase, the MDA levels increased significantly by 46%, 151.47%, and 368.38% compared to the levels observed in the Control. Similarly, the percentage increases observed compared to Control for the third treatment phases were 18.55%, 23.39% and 33.87% for 10mg/kg, 20mg/kg, and 30mg/kg K₂Cr₂O₇ respectively.

In the Dox groups (5 and 6), the Rats treated indicated similar spike increases (p < 0.05) in the levels of MDA compared to the levels observed in the Control. The percentage increases were 3.33% and 40% (First Phase), 2.21% and 27.94%(Second phase), and 26.61% and 28.23% (Third Phase) for Rats treated with 15mg/kg and 20mg/kg Dox treated groups respectively.

3.4 Rats treated with different doses of K₂Cr₂O₇ and Dox showed increased Cardiac markers (AST, LDH, and CK-MB) activities in the first treatment phase.

Results presented in Fig. 3 (A, B and C) show that rats exposed to K₂Cr₂O₇ and Dox respectively elicited a significant (p < 0.05) increase in AST, LDH, and CK-MB activities in the treatment phases relative to the control (Group 1).

In the phase 1, AST results indicated 22.53%, 37.56%, and 64.39% increases in enzyme activities. LDH activities also increased by 55.24%, 73.82%, and 116.10%. Also, CK-MB activities followed the increasing trend by 16.46%, 45.60%, and 87.63% for Rats treated with 10mg/kg, 20mg/kg, and 30mg/kg K₂Cr₂O_7, respectively, when compared to the Control group. Similarly, the rats treated with 15mg/kg and 20mg/kg Dox in groups 5 and 6 indicated increased AST, LDH, and CK-MB activities. The percentage increases in activities for groups 5 and 6 compared to the control group were 2.65% and 4.24% (AST), 0.23% and 18.33%(LDH), and 34.4% and 12.55%(CK-MB).

In the second phase, the AST results indicated 30.76%, 38.73%, and 93.56% increases in enzyme activities. LDH activities also increased by 34.37%, 47.17%, and 70.25%. Also, CK-MB activities followed the increasing trend by 33.15%, 58.80%, and 101.12% for Rats treated with 10mg/kg, 20mg/kg, and 30mg/kg K₂Cr₂O_7, respectively, when compared to the Normal group. Likewise, the rats treated with 15mg/kg and 20mg/kg Doxorubicin(Dox) in groups 5 and 6, respectively, indicated increases in AST, LDH, and CK-MB activities. The percentage increases in activities for groups 5 and 6 compared to the control group were 5.28% and 4.56%(AST), 38.33% and 56.43%(LDH), and 9.71% and 18.13%(CK-MB).

In the third phase, the AST results indicated 12.35%, 25.20%, and 33.89% increases in enzyme activities. LDH activities also increased by 3.56%, 10.32%, and 13.48%. Also, CK-MB activities followed the increasing trend by 8.55%, 25.48%, and 101.12% for Rats treated with 10mg/kg, 20mg/kg, and 30mg/kg K₂Cr₂O_7, respectively, when compared to the Normal group.

Similarly, the rats treated with 15mg/kg and 20mg/kg Doxorubicin (Dox) in groups 5 and 6, respectively, showed elevated levels of AST, LDH, and CK-MB activities. The percentage increases in activities for groups 5 and 6 compared to the control group were 5.28% and 4.56% (AST), 38.33% and 56.43%(LDH), and 9.71% and 18.13%(CK-MB).

3.5 Rats treated with different doses of K₂Cr₂O₇ and Dox increased Cardiac Troponin-I(CTnI) levels in all the treatment phases(First, Second and Third Phases).

The results in Fig. 4 shows the levels of CTnI investigated in the hearts of the Control, K₂Cr₂O_7, and Dox₋treated Rats with a significant increase in CTnI levels in all the treatment Phases. The percentage increase in CTnI levels for the Rats treated with 10mg/kg, 20mg/kg, and 30mg/kg K₂Cr₂O₇ compared to the Normal Control was 29.18%, 35.01%, 38.91% for the first phases.

In the second phase, the CTnI levels increased significantly by 63.76%, 65.07%, and 71.62% compared to the levels observed in the Control. Also, the percentage increases observed compared to Control for the third treatment phase were 2.28%, 3.33% and 9.52% for 10mg/kg, 20mg/kg, and 30mg/kg K₂Cr₂O₇ respectively.

In the Dox groups(5 and 6), the Rats treated indicated similar increases(p < 0.05) in the levels of CTnI compared to the levels observed in the Control. The percentage increases were 4.67% and 15.57% (First Phase), 20.09% and 10.48%(Second phase), and 3.81% and 4.77% (Third phase) for Rats treated with 15mg/kg and 20mg/kg Dox treated groups respectively.

3.6 Rats treated with different doses of K ₂ Cr ₂ O ₇ and Dox indicated increased C-Reactive Proteins (CRP) in all the treatment phases (First, Second and Third Phases).

Results presented in Fig. 5 shows spikes in C- Reactive Protein levels compared to Control, K₂Cr₂O_7, and Dox treated Rats. The percentage increase in CRP levels for the Rats treated with 10mg/kg, 20mg/kg and 30mg/kg K₂Cr₂O₇ compared to the normal Control were 35.62%, 45.21%, 82.19% for the first phases. In the second phase, the CRP levels increased significantly by 113%, 229%, and 300% compared to the observed control values.

Also, the percentage increases observed compared to Control for the third treatment phases were 2.56%, 9.29% and 18.27% for 10mg/kg, 20mg/kg, and 30mg/kg K₂Cr₂O₇ respectively. In the Dox groups(5 and 6), the Rats treated indicated similar spike increases(p < 0.05) in the levels of CRP compared to the levels observed in the Control. The percentage increases were 23.28% and 23.29% (First Phase), 38% and 114%(Second phase), and 2.56% and 7.05% (Third phase) for Rats treated with 15mg/kg and 20mg/kg Dox treated groups respectively.

3.7 Different doses of K₂Cr₂O₇ and Dox inhibited Nitric Oxide levels in the heart of treated Rats in all phases(First, Second and Third Phases).

Results presented on Fig. 6 shows decreases in the levels of NO availability in the hearts of K₂Cr₂O₇ and Dox-treated rats compared to the Control. The percentage decrease in NO levels for the Rats treated with 10mg/kg, 20mg/kg, and 30mg/kg K₂Cr₂O₇ compared to the normal Control were 4.65%, 18.61%, 27.91% for the first phase. Conversely, in the second phase, NO indicated an increase of 31.7%, 41.7%, and 76.8% compared to the Control.

Also, percentage increases were observed compared to control for the third treatment phases were as follows: 385%, 885% and 1200% for 10mg/kg, 20mg/kg, and 30mg/kg K₂Cr₂O_7, respectively. The NO levels in Dox groups (5 and 6) in the first phase indicated a slight decrease compared to control levels. In contrast, a significant increase (p < 0.05) was observed in the levels of NO in the second and third phases compared to the Control. The percentage decreases were 2.31% and 4.65% (First Phase), while the percentage increase was 19.5% and 1.22% (Second phase), and 750% and 1115% (Third Phase) for Rats treated with 15mg/kg and 20mg/kg Dox treated groups respectively.

3.8: The effect of administering K₂Cr₂O₇ and Doxorubicin intraperitoneal injection on the myocardial histoarchitecture of animals treated in the first phase (H&E X400) representative photomicrographs followed by light microscopy are indicated as follows:

Histopathology result shows Alphabeths a-f representing various photomicrographs of the treatment groups in phase one (21 days). (a) depicts Control rats that received an equal volume of normal drinking water orally for 21 consecutive days, (b) Rats that received oral administration of 10 mg/kg body weight of K₂Cr₂O₇ [Cr(VI)] compound for 21 consecutive days, (c) Rats that received oral administration of 20 mg/kg body weight of K₂Cr₂O₇ [Cr(VI)] compound for 21 consecutive days, (d) Rats that received oral administration of 30 mg/kg body weight of K₂Cr₂O₇ [Cr(VI)] compound for 21 consecutive days, (e)15 mg/kg body weight doxorubicin (2 days prior to sacrifice) administered intraperitoneally,(f)20 mg/kg body weight doxorubicin (2 days prior to sacrifice) administered intraperitoneally.

3.9 Effect of K₂Cr₂O₇ and Doxorubicin in phase 2 histopthology results H and E X400, representative photomicrographs followed by light microscopy are indicated as follows:

Fig 8 shows Alphabeths g-l representing various photomicrographs of the treatment groups in phase one(60 days). (g) Control rats that received an equal volume of normal drinking water orally for 60 consecutive days, (h) rats that received oral administration of 10 mg/kg body weight of K₂Cr₂O₇ [Cr(VI)] compound for 60 consecutive days, (i) Rats that received oral administration of 20 mg/kg body weight of K₂Cr₂O₇ [Cr(VI)] compound for 60 consecutive days, (j) Rats that received oral administration of 30 mg/kg body weight of K₂Cr₂O₇ [Cr(VI)] compound for 60 consecutive days, (k) Rats given 15 mg/kg body weight doxorubicin (2 days prior to sacrifice) administered intraperitoneally,(l) rats given 20 mg/kg body weight doxorubicin (2 days prior to sacrifice) administered intraperitoneally.

3.10: Effect of K₂Cr₂O₇ and Doxorubicin in phase 3 on the myocardial histoarchitecture of animals treated in the Third phase using the H&E staining E X400, representative photomicrographs followed by light microscopy are indicated as follows:

The effect of treatment groups was represented using Alphabeths m-r on Fig. 9 shows m Control rats fed a standard pellet diet and received an equal volume of distilled water, n shows Rats given 0.01% of K₂Cr₂O₇ [Cr(VI)] compound in feed, i.e., 10mg/kg of K₂Cr₂O₇ [Cr(VI)] compound per Kg Bodyweight in 100g of Rat feed for 60 days, o shows Rats given 0.02% of K₂Cr₂O₇ [Cr(VI)] compound in feed, i.e., 20mg/kg of K₂Cr₂O₇ [Cr(VI)] compound per Kg Bodyweight in 100g of Rat feed for 60 days, p shows Rats given 0.03% of K₂Cr₂O₇ [Cr(VI)] compound in feed, i.e., 30mg/kg of K₂Cr₂O₇ [Cr(VI)] compound per kg Bodyweight in 100g of Rat feed for 60 days, q shows Rats given 15 mg/kg body weight Dox (2 days prior to sacrifice) administered intraperitoneally, r shows Rats given 20 mg/kg body weight doxorubicin (2 days prior to sacrifice) administered intraperitoneally.

Many research data indicate that environmental exposure to Toxic metals is implicated in the severe pathogenesis of chronic diseases. In this present study, there was no mortality during the treatment phases. The rats also showed increased final body weights across groups, as seen in Tables 1. However, we observed a statistically marked increase in body weight of the control groups compared to those that received K₂Cr₂O₇ and Dox. There was also a significant difference in the relative heart weight of rats fed K₂Cr₂O₇ compared to the absolute heart weight of the Control and Dox-treated rats. It was also noted that as the concentration of K₂Cr₂O₇ administered increased, reduced relative heart weight was observed in the experimental Rats. K₂Cr₂O₇ intoxication may have been responsible for the rats' observed relative heart weight reduction. This finding agrees with (Soudani et al., 2011), where severe toxicity to brush border enzymes was observed by K₂Cr₂O₇ intoxication. Likewise, (Hayyan et al., 2016) and (Wuri et al., 2022) have also buttressed K₂Cr₂O₇ toxicity in kidneys and oocytes of exposed rats suggesting that oxidative damage may be responsible for the reduced relative organ weight.

Table 1

Effect of K₂Cr₂O₇ and Doxorubicin on the body weight, absolute and relative heart weights of rats (g).
		Heart Weight		Body Weight
		Absolute	Relative	Final	Initial
Control	Acute exposure	0.52 ± 0.03	0.36 ± 0.04	145.69 ± 1.42	131.4 ± 1.51
	Chronic Exposure	0.57 ± 0.03	0.33 ± 0.06	168.73 ± 1.48	126.4 ± 1.75
	Chronic exposure in food.	0.54 ± 01	0.34 ± 0.02	161.73 ± 0.03	133.40 ± 0.03
10mg/kg Cr	Acute exposure	0.51 ± 0.05	0.36 ± 0.02	140.80 ± 1.52	127.25 ± 1.32
	Chronic Exposure	0.58 ± 0.05	0.37 ± 0.02	156.80 ± 2.66	138.25 ± 1.84
	Chronic exposure in food.	0.60 ± 0.01	0.38 ± 0.01	156.73 ± 0.02	132.28 ± 0.02
20mg/kg Cr	Acute exposure	0.53 ± 0.10	0.37 ± 0.02	141.75 ± 3.50	121.75 ± 2.91
	Chronic Exposure	0.55 ± 0.10	0.35 ± 0.02	155.75 ± 3.56	137.75 ± 3.58
	Chronic exposure in food.	0.47 ± 0.03	0.29 ± 0.01	161.43 ± 0.02	132.55 ± 0.01
30mg/kg Cr	Acute exposure	0.54 ± 0.12	0.38 ± 0.01	141.23 ± 2.51	130.75 ± 0.80
	Chronic Exposure	0.59 ± 0.12	0.38 ± 0.01	153.23 ± 3.97	135.75 ± 0.85
	Chronic exposure in food.	0.59 ± 0.01	0.37 ± 0.12	160.25 ± 0.03	136.20 ± 0.02
15mg/kg dox	Acute exposure	0.53 ± 0.06	0.37 ± 0.02	144.50 ± 2.00	122.40 ± 1.14
	Chronic Exposure	0.58 ± 0.06	0.38 ± 0.01	159.50 ± 2.73	126.40 ± 1.24
	Chronic exposure in food.	0.47 ± 0.02	0.30 ± 0.13	156.43 ± 0.02	135.70 ± 0.02
20mg/kg dox	Acute exposure	0.55 ± 0.07	0.38 ± 0.01	143.5 ± 2.17	120.10 ± 1.20
	Chronic Exposure	0.57 ± 0.07	0.35 ± 0.01	163.5 ± 2.15	124.00 ± 1.68
	Chronic exposure in food.	0.47 ± 0.01	0.30 ± 0.01	156.15 ± 0.03	132.83 ± 0.01
Each value represents the mean ± SE; Values are significantly different at P < 0.05.

It was also deduced in this study that the Cr6 + exposure through the administration of K₂Cr₂O₇ in all the treatment phases induced oxidative damage to the heart, as evident in the decrease of antioxidants activities (SOD, CAT, and Gpx). The antioxidant enzymes assessed in this study, as seen in Fig. 1 (A), revealed a statistically marked decrease in the antioxidant activities of GPx, SOD and CAT in phase 1 compared to the Normal Control. Also, animal groups treated with Dox showed a significant decrease in GPx, SOD, and CAT compared to the control group. These enzymatic antioxidants are the primary defence blocks that mop out the toxicity effect of the overwhelming oxidant production arising from K₂Cr₂O₆ and Dox exposure. The weak antioxidant defences in the heart make it a porous target compared to other organs (Abarikwu et al., 2016; Boriskin et al.,2020; Ankita et al., 2019 Sharifi-Rad et al.,2020). Hence, SOD, CAT, and GPx activities are impaired, resulting in cellular lipids, proteins, and DNA damage and the onset of many chronic diseases. Also, the trend of Oxidant production was observed to be on the increase as the concentrations of both K₂Cr₂O₆ and doxorubicin treatments increased, resulting in a decrease in the antioxidant enzyme activities (GPx, CAT, and SOD) due to the overwhelming burden of the toxic agents (Boriskin et al.,2020; Wuri et al.2022).

In Phase 2, the antioxidant enzymes shown in Fig. 1 (B) indicated a similar trend in the decreased SOD, CAT, and GPx activities of the rats exposed to different doses of K₂Cr₂O₇ and Dox for 60days. The percentage decrease in the enzyme activities was higher than the decrease observed in the first treatment phase. This may result from the longer duration of the animal exposure to K₂Cr₂O₇, compromising the antioxidant defence system and resulting in cardio-damages (Krishnamurthy et al.,2015; Abarikwu et al., 2016; Ankita et al.,2019; Sharifi-Rad et al.,2020; Fagundes et al.,2022).

Similarly, Phase 3 presented in Fig. 1 (C) shows a decrease in the antioxidant activities of GPx, SOD and CAT in rats treated with different doses of K₂Cr₂O₇ supplemented in diets and Dox for 60days. The percentage decrease in antioxidant enzymes observed in rats in the second phase of treatment by oral gavage administration was significantly higher than in the third phase by diet supplementation (Foster et al.,2015). Our findings agree with (Foster et al.,2015), whose results propose that bolus gavage administration of manganese could be associated with higher brain manganese levels and, consequently, a higher risk of neurotoxicity. Therefore, this present study suggests that the higher cardiotoxicity effect observed in the oral Gavage- K₂Cr₂O₇ treated rats compared to the supplemented diet- K₂Cr₂O₇ treated rats may be responsible for the higher decreased antioxidant activities. These observations have raised concerns regarding the fitness of oral treatment administration using Gavage against diet supplementation due to the endocrine disruptor effects observed by the gavage method (Vandenberg et al., 2014; Foster et al.,2015).

Secondly, the increased MDA levels observed in all the phases in this present study, as evident in Fig. 2, further reiterate that Rats' treatment with different doses of K₂Cr₂O₇ and Dox induced oxidative stress. The prevalence of MDA in Cr(VI) treated rats is attributable to lipid peroxidation and an increase in ROS production induced by the K₂Cr₂O₇ treatments, thus degrading polyunsaturated lipids or fatty acids in the rats' perturbed biological system; hence may be responsible for the increased levels of MDA observed in this study (Farrokhian et al., 2019; Anam & Purwanto,2020).Also, Dox has proven to generate cardio-dysfunctions through the induction of free radicals such as oxyanions, hydroxyl, and hydrogen peroxide (Teraoka et al., 2000; Bhardwa et al., 2011; Hardik et al., 2013). Hence, increased MDA levels were observed.

Researchers have also elucidated the elevation of Cardiac markers such as AST, LDH, CK–MB used as indicators of myocardial tissue damage. These includes ischemia and infarction (Wagner,2003; Yang et al., 2014; Fernandez-Chas et al., 2018; Al-Malky et al.,2019, Lakhani et al.,2021; Firoozichahak et al., 2022). Figure 3 (A) in this present study indicated the Cardiotoxicity of K₂Cr₂O₇ and Dox exposure as evidenced in the increased LDH, AST, and CK-MB activities in the phase 1 compared to the control rats. The increased levels observed may be associated with severe Cardiotoxic symptoms such as chest pain, hypertension, decreased heart function, myocarditis, cardiomyopathy, shortness of breath, arrhythmias, palpitations, vascular disease, acute coronary syndrome, oedema, and vasculitis (Gaze 2007; Ganatra et al.,2019; Mehta et al., Patel et al.,2021; Firoozichahak et al., 2022). Similar results were also observed in our findings in both the second treatment phase (Fig. 3 (B)) and the Third treatment phase (Fig. 3 (C). There was a significant percentage increase in LDH, AST and CK-MB activities of the K₂Cr₂O₇ treated rats in the gavage-treated and diet-supplemented rats relative to the control group. However, the percentage increase was higher in the former, probably indicating that longer treatment with different doses of K₂Cr₂O₇ and Dox exposed the Rats to progressive Cardiotoxicity. The higher increase observed in the myocardial markers may be due to the mode of treatment administration, which suggested severe cardiotoxicity induction through gavage administration (Foster et al.,2015; Zhai et al.,2019).

Furthermore, CTnI, a quintessential and highly sensitive cardio-biomarker consisting of regulatory proteins controlling the calcium-mediated interaction of myosin and actin during myocyte contraction (Park et al., 2017; Chen et al.,2019, Chaulin, 2021). Figure 4 in this present study indicates a higher percentage increase in levels of CTnI in all the treatment phases relative to the control groups. The percentage increase progressed with increased duration of the K₂Cr₂O₇ exposure, i.e., 21days and 60days, respectively, compared to the control rats. The Dox groups similarly showed an increase in CTnI, indicating that both K₂Cr₂O₇ and Dox may be implicated in a dose and duration-dependent cardiotoxicity. CTnI changes in concentration levels are useful in diagnosing heart failure and other related cardiac diseases (Magnussen & Blankenberg., 2018). Clinical evidence has shown that patients who are often readmitted due to heart failure are those with persistent CTnI increases (Magnussen & Blankenberg., 2018).

Similarly, CRP levels in this present study, as shown in Fig. 5, indicated spikes in its levels in K₂Cr₂O₇ and Dox-treated rats in all the phases compared to the control groups. However,the percentage increases were significantly elevated in the second and third phases, possibly due to the longer treatment duration compared to the first phase. CRP are pro-inflammatory markers produced by the liver and used clinically to diagnose heart failure. Research pieces of evidence support that there may have occurred intestinal hypoperfusion due to the translocation of K₂Cr₂O₇ oral administration from the stomach into the bloodstream resulting in vascular inflammation and subsequent increased levels of CRP observed in this study (Anand et al., 2005; Magnussen & Blankenberg, 2018; Vulesevic et al.,2019).

Also, in this present study, Fig. 6 indicated significant decreases in the levels of NO availability in the hearts of the K₂Cr₂O₇ and Dox treated Rats in the first phase compared to the normal Control. In contrast, an increase was observed in the second and third treatment phases. Many research findings has stated that NO is synthesized by a family of nitric oxide synthases with three isoforms, which include inducible (iNOS), neuronal (nNOS), and endothelial NOS (eNOS). While iNOS is upregulated in activated macrophages, nNOS is primarily expressed in the neural tissue, and eNOS abounds in the endothelium, diffuses to the smooth muscles and enables the endothelial vascular homeostasis, which controls dilation or constriction through the activation of soluble guanylyl cyclase-cGMP-dependent protein kinase pathway. In addition, the impairment of NO synthesis is pertinent in the diagnosis of chronic heart failure, coronary artery diseases, arteriosclerosis involving stable and unstable angina and myocardial infarction hypertension, and as well pulmonary arterial hypertension, which progresses to the right ventricle failure ( Ferlito, 2000; Silva-Costa-Gomes; Dunham-Snary et al. 2017; Frump et al., 2018; Kurakula et al.,2021 Finkelman et al.,2017; Tran et al.,2022). The discrepancy observed in NO levels in all treatment phases compared to the normal Control may be due to an overexpression of iNOS, whose induction occurs primarily in conjunction with infection and inflammation as part of the defence response. iNOS expression is minimal under normal conditions; however, it is upregulated in response to pro-inflammatory cytokines and thus generates much more NO than eNOS does. Hence, High NO levels may have occurred in response to inflammation due to K₂Cr₂O₇ exposure for 60 days, while the decrease in NO levels in the groups treated with K₂Cr₂O₇ and Dox may have resulted from the inhibition of eNOS expression due to treatment exposure for 21 days. Thus, signalling cardiotoxicity owing to the development of systemic and pulmonary hypertension, which plays a key role in the pathophysiology of heart-related ailments (Wu et al., 2021). Furthermore, the increase in NO levels in the DOx treated groups as seen in this study, especially in the second and third phases, indicates an upregulation of nitric oxide synthase activities as observed by other researchers leading to an increase in the concentration of nitric oxide, thereby enhancing the production of the highly toxic peroxynitrite (Wu et al., 2021).

A report by Lee et al.(2016) has shown that NOS can play compensatory roles in laboratory animals with one or two disrupted isoforms. For example, the up-regulation of nNOS expression compensated the non-expression of eNOS and vice versa. These strides promises a useful research tool to enhance a discreet interpretation of discrepancies in NO availability and disease prognosis.

The histopathological examination in Fig. 7 shows the Effect of K₂Cr₂O₇ treatment in phase 1, revealing control rats(a) as having the normal histoarchitecture of the heart, as the myocardium showed a rich network of vasculature, capillaries, nucleus, and pericytes. In contrast, the groups treated with K₂Cr₂O₇ (b, c and d) indicated areas of mild tissue oedema due to treatments exposure suggesting the pathogenesis of lung oedema due to K₂Cr₂O₇ induced Cardiotoxicity resulting from pulmonary hypertension from hypoxic pulmonary vasoconstriction induced by Cr(VI) agents resulting in congestive heart failure ( Dunham-Snary et al. 2017). The Dox groups (e and f) showed a higher degree of cardiac damage as there was myotocytolysis and hyaline necrosis in addition to pulmonary oedema, the affected cells' loss of striation, fragmentation of the myocytes, intense eosinophilic staining of the sarcoplasm (myocytes cytoplasm) with nuclear pyknosis and karyorrhexis. In group 6, oedema seemed to be more severe and widespread.

Similarly, Figs. 8 and 9 revealed the histopathology examination of K₂Cr₂O₇ and Dox treated Rats in phase 2 and 3 showing Control groups (g and m, respectively) expressed a normal heart structure while (h, I, j) and (n, o, p) indicated areas of myocyte displaced, indicating pulmonary oedema; Dox groups (k and l) and (q and r) similarly showed areas of pulmonary oedema, hyaline necrosis and myocytolysis for the second and third study, respectively. Pulmonary oedema has been of interest to cardiologists, and pulmonologists, due to its prevalence in the onset of heart diseases.

Pulmonary oedema is induced by the movement of fluid extravascularly into the interstitium and alveoli of the lungs. The most common causes of pulmonary oedema are Cardiomyopathy, Aortic valve disease, coronary heart disease, hypertension, mitral valve disease,` and congestive heart disease. Other causes include damage to the lungs, kidneys, pancreas, drugs, trauma, and toxins toxins (Ingbar, D. H.,2019; Logan et al.,2019., Barile, 2020 Moreno-Gonzalez et al.,2022).. Furthermore, Many researchers categorized pulmonary oedema into permeability oedema (with and without diffuse alveolar damage (DAD), hydrostatic pressure oedema, and mixed oedema, where there is both an increase in membrane permeability and hydrostatic pressure (Paone et al.,2018; Zompatori et al., 2014; Barile, 2020; Matthay et al., 2019; Carlicchi et al., 2021; Ingbar, D. H. (2019). However, it is not clear which physiologic category of the pulmonary oedema observed in this study due to the K₂Cr₂O₇ treatments.

Thus, this study demonstrated that Cr [VI] compared well with Dox in cardiotoxicity induction with accompanying oxidative stress, inflammatory and histo-hepatic responses in the rats. The dose and duration of treatments for the biochemical and myocardial indices are not dependent on body weight alone, hence may need pharmacokinetic modifications for conversion between animals and humans. Also, the mechanism that resulted in pulmonary oedema regarding the myocardial indices observed in this study is unclear warranting further studies for clarification.

Funding

The authors did not receive support from any organization for the submitted work.

Financial interests

The authors declare they have no financial interests.

Competing Interest

The authors have no competing interests to declare that are relevant to the content of this article.

Ethics approval and consent to participate

All investigations involving the experimental animals adhered strictly to the guidelines for the care and use of animals as defined by the National Institute of Health Guide for the Care and Use of Laboratory Animals. Committee on Care and Use of Laboratory Animals, US Department of Health and Human Services, Public Health Service, Institute of Laboratory Animal Resources, USA. The experiment protocols were performed after the ethical approval was granted by the College of Natural Science, Research Ethics Committee (CREC/002/21), Michael Okpara University of Agriculture.

Consent for publication

Not applicable

Availability of data and material

All data generated or analyzed during this study are included in this published article. Any further reasonable request can be made through the corresponding author.

Authors' contributions

Conceptualization: Onyedikachi, Uchechi Bliss, Awah, Favour Matthew; Kpobari Nkpaa, Methodology: Onyedikachi Uchechi Bliss, Kpobari Nkpaa, Egbuonu Anthony Cemaluk Chinedum; Awah Favour Matthew Formal analysis and investigation: Onyedikachi,Uchechi Bliss, Awah Favour Matthew; Writing - original draft preparation: Onyedikachi Uchechi Bliss; Awah Favour Matthew.; Writing - review and editing: Onyedikachi Uchechi Bliss; Ijioma,Solomon Nnah ;Egbuonu, Anthony Cemaluk Chinedum; Resources: Onyedikachi,Uchechi Bliss; Awah, Favour Matthew.; Supervision: Egbuonu, Anthony Cemaluk Chinedum, Ijioma, Solomon Nnah.

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Comparative Evaluation of Vehicle, Dose, and Duration-related Oxidative, Cardiotoxic, Inflammatory and Histologic Responses of Chromium 6⁺ and Doxorubicin in Rats’ Heart

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Abstract

Figures

1.0 Introduction

2.0 Material And Methods

2.1 Chemicals and Drugs

2.2 Animal Husbandry

2.3. Experimental Design

2.4 Preparation of heart samples

2.4.1 Body Weight and Relative Organ Weight

2.5 Biochemistry Assay

2.6 Immuno Assays

2.7 Preparation of slides for histopathological evaluation

2.8 Statistical Analysis

3.0 Result

4.0 Discussion

Conclusion

Declarations

References

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