ANTI-TOXIC PRINCIPLES FROM MUSA SAPIENTUM AND MORINGA OLEIFERA AMELIORATED SKIN HISTO-PATHOLOGY, DECREASED LIPID PEROXIDATION AND PROMOTED MELATONIN/TNF-ALPHA/ P53-MEDIATED APOPTOSIS IN CADMIUM CHLORIDE-INDUCED CARCINOGENESIS IN RATS

Anti-toxic

The WHO listed cadmium as one of the 10 chemicals or groups of chemicals of concern for human health (Andjelkovic et al. 2019).The liver and the kidney are the major deposit sites of cadmium, receiving approximately 30% of cadmium deposits.The remaining cadmium deposits are distributed across body systems leading to systemic dysfunctions such as skin alopecia and ulceration (Lansdown et al. 2001), inflammation and hepato-toxicity (Bernhoft 2013;Wang and Du 2013;Angjelkovic et al. 2019).Cadmiuminduced carcinogenesis in animal-models are similar in features to those of human cancers, making it a reliable model for evaluating anticancer drugs (Wang and Du 2013;Angjelkovic et al. 2019).
Lipid peroxidation is implicated in inflammation and carcinogenesis due to its causative production of mutagenic reactive aldehydes such as malondialdehyde, defective cell membrane and cellular damage (Akinlolu et al. 2012).Melatonin which is majorly metabolised in the liver (Mohammadi et al. 2016) is an established antioxidant, antiinflammatory and anti-cancer candidate (Akinlolu et al. 2012;Lampiao and Du Plessis 2013;Zamfir et al. 2014;Mohammadi et al. 2016).This makes melatonin to be of importance in the treatments of hepatic oxidative stress and carcinogenesis.In addition, tumour necrosis factor-alpha (TNFa) is a pro-inflammatory cytokine that plays inductive roles in immuneand inflammatory-responses, and cancer-associated chronic inflammation across body systems (Liu et al. 2004;Zahr et al. 2010;Chu 2013).Furthermore, p53 which is usually referred to as the guardian of the genome is involved in the induction of apoptotic and cell cycle arrest genes when the body system is exposed to inflammation, DNA damage and carcinogenesis (Chang et al. 2010;Toshinori and Akira 2011;Xiao et al. 2013).
The skin is the most external and first protective body layer (Moore et al., 2014).The liver, on the other hand impact strongly on systemic processes such as drug metabolism, drug detoxification and efficient systemic functioning of the body (Moore et al. 2014).Cadmium usually exists as a divalent cation, complexed with other elements, such as cadmium chloride (CdCl 2 ) (Bernhoft 2013;Angjelkovic et al. 2019).Therefore, in order to further determine the anticancer potentials of MSF1 (isolated from MS sucker) and MOF6 (isolated from MO leaves), this study evaluated the effects of MSF1 and MOF6 on skin histology, hepatic oxidative stress, and immuno-modulations of melatonin, TNFa and p53 proteins in CdCl 2 -induced carcinogenesis in rats.

Ethical Approval
The ethical approval for this study was obtained from the Ethical Review Committee of the University of Ilorin, Nigeria via Ethical Approval number: UERC/ASN/2018/1161.In addition, all experimental procedures of this research were carried out in compliance with the international guidelines for laboratory animal use and care such as the US guidelines (NIH publication #85-23, revised in 1985) and the European Community guidelines (EEC Directive of 1986; 86/609/EEC).

Authentication of MO Leaves and MS Suckers
MO leaves and MS suckers were obtained freshly from local forest reserves in Ilorin of Kwara State, Nigeria.The samples were identified and authenticated by a Botanist in the Department of Plant Biology, of University of Ilorin, Nigeria.Thereafter, the plant materials were deposited at the herbarium of the Department of Plant Biology, University of Ilorin.The obtained MS suckers and MO leaves were assigned Herbarium identification numbers: UILH/002/1182 and UILH/001/1249, respectively.

Preparations and Ethanolic Extractions of MO Leaves and MS Suckers
MO leaves and MS suckers were shade-dried at the laboratory unit of the Department of Chemistry, University of Ilorin, Nigeria.A total weight of 5.2 kg of MS suckers and 4.0 kg of MO leaves were pounded, powdered, extracted with distilled ethanol and concentrated with the aid of a rotary evaporator at 40°C-60°C.Ethanol was used to remove impurities while the extracted product was heated in a conical flask.The conical flask contained boiling chips/anti-bumping granules which were used to prevent liquid ethanol from 'bumping' into the condenser while flowing into a container.The procedure was continuously recycled to ensure that it keeps running.
Twenty-four hours later, the mixture was decanted and sieved.New distilled ethanol was added to the sieved MO leaves and MS suckers following decantation; and left for another 24 h.The procedure was stopped once the colour quality and texture of the dissolved MO leaves and MS suckers in ethanol became evidently low in comparison with previous decanted solutions.Column chromatography was carried out to obtain different fractions of MO leaves and MS suckers following ethanol-separation.

Animals Care and Feeding
Fifty-five male Wistar rats with an average weight of 200 g were acclimatised for 5 days in the animal house of Faculty of Basic Medical Sciences of University of Ilorin, Nigeria.Following acclimatisation, the rats were randomly divided into 11 groups (Control Group 1 and Experimental Groups 2-11) with five rats per group.Each group of rats was kept in a wire gauzed cage.All rats were kept under standard conditions and fed water and food ad libitum.Bodyweights (g) of all rats were measured on day 1 of experimental procedure and at the end of each week.

Induction of Carcinogenesis
A single dose of 1.25 mg/kg bodyweight CdCl 2 was injected into each rat of Experimental Groups 2-6 via intra-peritoneal administration on day 1 to induce carcinogenesis.Each rat was monitored daily from days 1-14.Induction of cancer (carcinogenesis) was confirmed after 14 days via morphological appearance of alopecia and ulcers on the skin of the rat.

Experimental Procedures and Drugs Administration
The 11 groups of rats employed in this study were categorised into six groups as below and as highlighted in Tables 1-4.
Baseline Control Group 1: Rats of Control Group 1 received only physiological saline for 56 days (days 1-56).
Negative or Toxic Control Group: Rats of Experimental Group 2 received only single intraperitoneal administration of 1.25 mg/kg bodyweight of CdCl 2 on day 1 but were left untreated following CdCl 2-induced skin carcinogenesis for 42 days (days 14-56).
Anti-Cancer Treatment Groups: Rats of Experimental Groups 3-5 received single intraperitoneal administration of 1.25 mg/kg bodyweight of CdCl 2 on day 1, and were posttreated with oral administrations of 15 mg/kg bodyweight of MOF6, 30 mg/kg bodyweight of MOF6 and 10 mg/kg bodyweight of MSF1, respectively, for 42 days (days 14-56).
Positive Control Group: Rats of Experimental Group 6 received single intraperitoneal administration of 1.25 mg/kg bodyweight of CdCl 2 on day 1, and were post-treated with intravenous injection of 0.5 mL/200 g of cisplatin and oral administration of 3.35 mg/kg bodyweight of doxorubicin for 14 days (days 15-28).Post-treatment with 0.5 mL/200 g of cisplatin and oral administration of 3.35 mg/kg bodyweight of doxorubicin was stopped on day 28, because the rats could not tolerate further treatment with cisplastin and doxorubicin beyond 14 days.
Toxicological Profiling Groups: Rats of Groups 7 and 8 received only oral administrations of 15 mg/kg and 30 mg/kg bodyweight of MOF6, respectively, for 56 days (days 1-56).Rats of Group 9 received only oral administration of 10 mg/kg bodyweight of MSF1 for 56 days (days 1-56).
Hepato-protective and Cancer Prevention Groups: Rats of Experimental Group 10 received single intraperitoneal administration of 1.25 mg/kg bodyweight of CdCl 2 on day 1 and were post-treated with oral administration of 30 mg/kg bodyweight of MOF6 for 56 days (from days 1 to 56).Rats of Experimental Group 11 received single intraperitoneal administration of 1.25 mg/kg bodyweight of CdCl 2 on day 1 and were post-treated with oral administration of 10 mg/kg bodyweight of MSF1 for 56 days (from days 1 to 56).Therefore, in-order to determine if extracts-doses could prevent CdCl 2 -induced carcinogenesis, there was no confirmation of induction of cancer in rats of Groups 10 and 11 as treatment with extracts-doses commenced on day 1 after intraperitoneal administration of CdCl 2 -dose.

Animal Sacrifice
All rats were sacrificed by cervical dislocation following completion of experimental procedures either on day 28 (Experimental Group 6) or on day 56 (Control Group 1 and Experimental Groups 2-5 and 7-11).

Histo-Pathological Evaluations of the Skin of Rats
The dorsal skin area of each rat in all Groups was scraped, resected and fixed in 10% formal saline of at least five times of its volume.The fixed skin tissues were prepared for light microscopy by employing standard histological methods.Tissue sections were stained via haematoxylin and eosin stain method as previously described (Akinlolu et al. 2017).

Evaluations of Lipid Peroxidation
The thiobarbituric acid assay method was employed for quantification of malondialdehyde levels in liver homogenates of all rats as previously described (Akinlolu et al. 2012).

Sera Melatonin, Sera TNFa and Liver Tissues' p53 Proteins Concentrations Using Enzyme Linked Immunosorbent Assay (ELISA)
The thoracic cavity of each rat was exposed and 5 mL blood sample collected via the ventricles of the heart into lithium heparinised bottles.The blood samples were centrifuged to obtain the sera.Thereafter, the sera were used for quantitative histochemical enzyme linked immunosorbent assays (ELISA) of levels of melatonin and TNFa proteins in rats of Groups 1-11.
In addition, liver tissues were isolated immediately following animal sacrifice and thoroughly homogenised with the aid of porcelain mortar and pestle in ice-cold 0.25 M sucrose, in the ratio of 1 g to 4 mL of 0.25 M sucrose solution.Skin and liver tissue homogenates were filled up to 5 mL with additional sucrose and collected in a 5 mL serum bottle.The resulting homogenates were centrifuged at 3,000 revolutions per minute for 15 min using a centrifuge (Model 90-1).Supernatants were obtained from the liver tissue homogenates using pasteur pipettes and stored in a freezer at −20°C.Thereafter, quantitative histochemical ELISA of levels of p53 protein in obtained supernatants of rats of Groups 1-11 were conducted.Obtained data acquired from the micro-plate ELISA results were recorded and computed.

Statistical Analyses
Statistical analyses of data and comparisons between groups were conducted for any significant difference using one-way analysis of variance (ANOVA).Tukey post-hoc test was further employed for groups' comparisons.Statistical level of significance was set at p ≤ 0.05.

Histo-Pathological Evaluations of the Skin of Rats
Morphological observations showed dorsal skin ulceration in rats of CdCl 2 -treated Group 2 (Figure 1), compared with normal skin morphology in rats of Control Group 1; and normal skin morphology with minimal distortions in .Histo-pathological evaluations of the skin showed poorly outlined and thinner epidermis, hypo-dense dermis with sparse hair follicles and non-distinct hypodermis in rats of Group 2, when compared with Control Group 1 (Figures 2 and 3).

Malondialdehyde Levels in Liver Homogenates of Rats
Results showed no significant difference (p ≥ 0.05) of malondialdehyde levels in rats of Group 2, when compared with Control Group 1 and Groups 3-11 (Table 1).

Sera Melatonin Levels in Rats
Results showed no significant difference of melatonin levels (p ≥ 0.05) in Group 2 when compared with Control Group 1 and Groups 3-11 (Table 2).

Sera TNFa Levels in Rats
Results showed no significant difference of TNFa levels (p ≥ 0.05) in rats of Group 2, compared with Control Group 1 and Groups 3-6 and 8-11 (Table 3).However, there was a pattern of lower TNFa in rats of Group 4 when compared to Group 2 although it is not statistically significant (Table 3).

Levels of p53 in Liver Homogenates of Rats
Results showed statistically significant higher (p ≤ 0.05) levels of p53 in rats of Experimental Group 2, when compared with Control Group 1 and Groups 3-8, 10 and 11 (Table 4).

DISCUSSION
The observed dorsal skin ulceration and histo-pathological anomalies of the skin of rats of CdCl 2 -treated Group 2 suggest that observed CdCl 2 -induced skin alopecia and ulcers are associated with inhibited wound healing, dystrophic hair papillae, depigmentation and defective hair root sheath.These findings are in agreement with those of Lansdown et al. (2001) which reported that treatment of surgically induced skin wound with 1% CdCl 2 did not restore and re-epithelialise skin ulceration in rats, and there were nonresolved swelling, abnormal epidermal cell growth, wound waste and sustained mass of inflammatory cell penetration.In addition, the observed dermal atrophy with sparse hair follicles in this study was possibly induced by decreased number of cellular elements, loss of intercellular substance and degeneration of fibrous structures as posited by Abraham and Roga (2014).Histo-pathological evaluations showed normal histo-architectures of the skin components, though with minimal distortions in rats of Groups 3-11, when compared with Control Group 1 (Figures 2 and 4-8).These observations indicate that anticancer post-treatments or chemo-preventive co-administrations with MOF6 high-dose and MSF1-dose ameliorated and prevented CdCl 2 -induced skin skin ulceration, histo-pathology and carcinogenesis.
Malondialdehyde is a by-product of lipid peroxidation.Thus, upregulation of tissue level of malondialdehyde indicates induction of un-restrained oxidative stress (Akinlolu et al. 2012).The computed higher levels but non-significant upregulation of malondialdehyde levels in rats of Group 2 when compared with Group 1 (Table 1) implies mild induction of oxidative stress following CdCl 2 -exposure in rats of Group 2.
Post-treatments of CdCl 2 -induced oxidative stress with MOF6 low-dose, MOF6 high-dose and MSF1-dose resulted in non-significant downregulations of malondialdehyde levels in rats of Groups 3-6, when compared with Group 2 (Table 1).In addition, the commencement of treatment of CdCl 2 -induced carcinogenesis with MOF6 high-dose and MSF1-dose on day 1 and continued from days 2 to 56 resulted in non-significant downregulations of malondialdehyde levels in Groups 10 and 11, when compared with Group 2 (Table 2).These observations implied that MSF1 and MOF6 did not offer significant antioxidant potentials against CdCl 2 -induced oxidative stress.
Melatonin is an established antioxidant which targets the mitochondria for induction of its antioxidant effects (Russel et al. 2016).Melatonin is, therefore, able to inhibit lipid peroxidation via its antioxidant activities (Lampiao and Du Plessis 2013;Zamfir et al. 2014;Mohammadi et al. 2016).Furthermore, tissue-inflammation is associated with and accompanied by production of reactive oxygen species and free radicals as well as carcinogenesis.Therefore, the anti-inflammatory effects of Melatonin increases its antioxidant capability (Lampiao and Du Plessis 2013;Zamfir et al. 2014;Mohammadi et al. 2016;Russel et al. 2016).Thus, upregulation of melatonin levels promotes resolutions of oxidative stress, DNA and cellular damage, inflammation and carcinogenesis.The nonsignificant downregulation of melatonin levels in rats of Group 2 when compared with Group 1 (Table 2) indicated that CdCl 2 -exposure led to mild depletion of the antioxidant system in rats of Group 2.
Post-treatments of CdCl 2 -induced reduction in the antioxidant system with doses of MSF1 and MOF6 resulted in non-significant upregulation of melatonin levels in rats of Groups 3-6, when compared with Group 2 (Table 2).Similarly, the commencement of treatment of CdCl 2 -induced carcinogenesis with MOF6 high-dose and MSF1-dose on day 1 and continued fromd 2 to 56 resulted in non-significant increased melatonin levels in Groups 10 and 11, when compared with Group 2 (Table 2).These observations implied that MSF1 and MOF6 did not offer significant pro-melatonin potentials against CdCl 2 -induced depletion of the antioxidant system.
Tissue-inflammation is associated with releases of pro-inflammatory cytokines such as TNFa (Liu et al. 2004;Zahr et al. 2010;Chu 2013).Hence, the defense system of the body ensures inhibition of upregulation of pro-inflammatory cytokines but promotion of upregulation of anti-inflammatory cytokines.Thus, the non-significant upregulation of TNFa levels in rats of Group 2 when compared with Group 1 (Table 3) indicated that CdCl 2 -exposure resulted in mild induction of cancer-associated inflammation in rats of Group 2.
Post-treatments of CdCl 2 -induced inflammation with MOF6 low-dose and MSF1dose resulted in non-significant downregulations of TNFa in rats of Groups 3 and 5, when compared with Group 2 (Table 3).Similarly, the co-administration of CdCl 2 with MOF6 high-dose and MSF1-dose on day 1, and further administrations of extracts only from days 2-56, resulted in non-significant downregulation of TNFa in rats of Groups 10 and 11, when compared with Group 2 (Table 3).Thus, MSF1 and MOF6 MSF1 and MOF6 did not offer significant anti-inflammatory potentials against CdCl 2 -induced inflammation.
Tumour protein p53 is a pro-apoptotic and tumour suppressor gene.Hence, p53 is referred to as the guardian of the genome.p53 exists at a low level in its quiescent normal state and in normal conditions.Contrariwise, p53-upregulation occurs in cancer-associated chronic inflammation, DNA/cellular damage and carcinogenesis (Chang et al. 2010;Toshinori and Akira 2011;Xiao et al. 2013).Once upregulated, p53 becomes converted into its active state in-order to implement the repairs of DNA and cellular damages resulting in induction of arrest of the cell cycle.Following successful DNA and cellular procedures, normal cell cycle resumes and p53 level goes back to its low level.
The reported significant upregulation of p53 levels in rats of Group 2 when compared with Control Group 1 (Table 4) indicated that CdCl 2 -exposure led to induction of DNA and cellular damage with accompanied carcinogenesis in rats of Group 2. The observed consistent upregulation of p53 levels in rats of Group 2 at the end of the 8-week experimental procedure indicated sustained CdCl 2 -induced carcinogenesis in the absence of cancer treatment.This condition promotes inhibition of the capacity of p53 to repair DNA and cellular damage ensuring that remediating roles of p53 are used up and leading to un-restrained carcinogenesis.This state of anomaly will aid survival of cancer cells and promote tumorigenesis.The observed CdCl 2 -induction of carcinogenesis and toxicity in this study are in agreement with those of previous studies which reported cadmium-induction of oxidative stress (Wang and Du 2013;Mohamed et al. 2019), inflammation (Liu et al. 2009;Bernhoft, 2013;Wang and Du 2013), apoptosis and carcinogenesis (Júnior et al. 2020).
Post-treatments of CdCl 2 -induced carcinogenesis with MOF6 low-dose, MOF6 high-dose and MSF1-dose resulted in significant downregulations of p53 levels in rats of Groups 3-5, when compared with Group 2 (Table 4).These results indicate that MSF1 and MOF6 have significant capacities to promote the DNA repair mechanism of p53 and have rapid and very significant pro-apoptotic, tumor suppression and anticancer effects ensuring the return of p53 to normal levels following fast amelioration of CdCl 2 -induced carcinogenesis.
The co-administrations of CdCl 2 with MOF6 high-dose and MSF1-dose on day 1, and further administrations of extracts only from days 2-56, resulted in downregulations of p53 in rats of Groups 10 and 11, when compared with Group 2 (Table 4).Thus, MSF1 and MOF6 conferred chemo-prevention, cyto-protective, pro-apoptotic and anticancer capacities against CdCl 2 -induced carcinogenesis in rats.
In addition, cancer stem cells (CSCs) are evasive and are not destroyed by different cancer treatment procedures due to their capacities to escape DNA repair mechanism of p53 (Chen et al. 2013;Plaks et al. 2015).Thus p53 evaluation is of relevance in the production of anticancer drugs which have the capacity to destroy CSCs.The noted significant anticancer effects of MOF6 and MSF1 on p53 levels in CdCl 2 -induced carcinogenesis indicate that these plant fractions possibly contain anti-cancer drug candidates which can specifically target CSCs.
Furthermore, the anticancer potentials of MSF1 and MOF6 on p53 levels observed in this study are as good as post-treatments with a combined administration of cisplatin and doxorubicin (Table 4).These findings indicate that MSF1 and MOF6 possibly possess anticancer potentials that merit further assessments for the developments of anticancer drug candidates that can specifically target CSCs.
The non-significant downregulations of malondialdehyde, melatonin and TNFa in rats post-treated with doses of MSF1 and MOF6 against CdCl 2 -induced oxidative stress and inflammation possibly imply that higher doses of these plant fractions or extension of treatment days may be required for significant effects to be elicited.

CONCLUSION
Overall, the observations of this study suggest that post-treatments of CdCl 2 -induced carcinogenesis with MOF6 low-dose, MOF6 high-dose and MSF1-dose ameliorated skin histo-pathology and resulted in significant downregulations of p53 levels.These observations indicate that MSF1 and MOF6 possibly possess cyto-and histo-protective, hepato-protective and anticancer potentials against CdCl 2 -induced carcinogenesis.Thus, MSF1 and MOF6 are recommended for further evaluation as anticancer drug candidates.

Figure 1 :
Figure 1: Photograph of skin of rat of Experimental Group 2, which received single intra-peritoneal administration of 1.25 mg/kg bodyweight CdCl 2 (day 1) only.Morphological observations show skin ulceration and visible hair loss.

Figure 2 :
Figure 2: Photomicrograph of skin of rat of Control Group 1, which received physiological saline from days 1-56.Histological observations show well outlined epidermis.The dermis is distinct and contains numerous and visible hair follicles.Haematoxylin and eosin stain × 120; Scale bar: 50 μm; E = epidermis; HF = hair follicle; D = dermis; H = hypodermis; SM = skeletal muscle.

Figure 3 :
Figure 3: Photomicrograph of skin of rat of Experimental Group 2, which received single intra-peritoneal administration of 1.25 mg/kg bodyweight of CdCl 2 (day 1) only.Histo-pathological observations show poorly outlined and thinner epidermis compared with Control Group 1.The dermis is hypo-dense with sparse hair follicles and the hypodermis appears not distinct.Haematoxylin and eosin stain × 120; Scale bar: 50 μm; E = epidermis; HF = hair follicle; D = dermis; H = hypodermis; SM = skeletal muscle.