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There is a growing understanding and acceptance of the concept that Reactive Oxygen Species (ROS), which are aetiological agents in the development of a range of diseases. Among these diseases is colorectal cancer [1]. Oxidative stress is an imbalance between production of ROS and the ability of cellular defense system to diminish ROS level and repair the damage caused by them [2].

Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth most common cause of death [3,4]. Almost invariably it presents as pathology caused by persistent oxidative stress and inflammation [5].

The plant families Cycadaceae are palm-like plants which have survived the Mesozoic era with little change [6]. Cycads are widely distributed in tropical and subtropical regions [7]. Evidence that crude cycad material was carcinogenic was obtained early in 1962 when rats fed cycas seed meal diet were killed because of palpable abdominal tumour masses, ascites, and a rapidly developing anaemia [8]. Previous studies by [9] found that rats fed crude cycad meal developed hepatocellular carcinomas as well as kidney and intestinal tumours. It was later found that the carcinogen in cycad is cycasin [10], which was first isolated from the seeds of C.revoluta Thumb, identified by [11] and later found by [12] in C. circinalis Linn.

Concentrations of cycasin are highest in the seeds and roots, but present in all parts of the plant [9]. Cycasin is toxic only when given orally, and its toxicity appears after a period of about 12 hours [8]. There are reports on the carcinogenicity of cycasin in rats[13-15] in mice [16-18] hamsters [19] guinea pigs [20], rabbits [21] and aquarium fish [22].

Cycasin is synthesized from Methylazoxymethanol (MAM) by the enzyme UDP-glucosyltransferase [23]. It is metabolized by plant, bacterial and mammalian B-glucosidases to liberate the aglycone, MAM. MAM is the active toxic metabolite of cycasin [13]. It spontaneously breaks down into reactive molecules such as methydiazonium ions and carbon-centered free radicals which can methylate DNA [24]. The accumulation of lesions in DNA in form of methylated sites is responsible for the teratogenic, mutagenic, hepatotoxic, carcinogenic and neurotoic properties of MAM [24].

Though, there is evidence of the carcinogenic capacity of cyacs leaf powder, when incorporated in diet (5% w/w) [25], there is dearth of information on the ability of the extract to induce oxidative stress and colon cancer in experimental animals. The present study was therefore designed to compare the oxidative stress and histopathological change-inducing capacity of C.circinalis leaf powder and the ethanolic leaf extract in rats.

Preparation of the ethanolic leaf extracts of Cycas circinalis

C.circinalis leaf powder (150 g) was soaked in 1.5 liters of absolute ethanol (99% w/v) for 72 hours with regular stirring. The extract was then filtered through a sintered funnel and Whatman No. 1 filter paper. The filtrate obtained was concentrated in a hot water bath at 70°C. The resultant greenish paste deposited at the bottom of the flask was then dried in an oven at 45°C. The dry extracts was then scraped with a stainless spatula, crushed in a mortar and then kept in glass bottle until required. The weight of extract obtained from 150 g of the pulverized dry leaf was estimated.

LD50 determination

The oral LD50 of the ethanolic leaf extract of C.circinalis was determined using the method of Lorke [26].

Animals grouping and treatment

The rats were randomly divided into seven (07) groups of 5 rats per each. Members of each group were housed separately in a clean, disinfected cage in a room with a 12-hour light/dark cycle. The rats were maintained on growers mash (Bendel Feed and Flour Mill (BFFM), Ewu, Nigeria) and water ad libitium for one week prior to commencement of the experiment. When the experiment started, group 1 rats were maintained on normal feed, growers mash without cycas leaf powder or the extract (control).

Group 2 rats were exposed to the leaf powder tainted growers mash (5% w/w) continuously for six weeks. Group 3 rats were placed on normal feed but administered ethanol extract of cycas leaf (10 mg/kg body weight) while groups 4,5,6 and 7 were exposed to the extract at a dose of 30,50,80 and 100 mg/kg body weight respectively once a week for 6 weeks by gavage. Cycas circinalis leaf extract was dissolved in 25% ethanol prior to administration. All groups were kept on their respective diet for six weeks.

Animal sacrifice

This study was carried out in strict compliance with the ethics in Guidelines and Specification on Experimental Animal Care [27]. At the end of the treatment period each rat was anesthetized using halothane saturated chamber.

Sample preparation

While under anaesthesia the abdominal region was opened and the colon was excised and sections obtained for histological examination and biochemical assessment of colon homogenate supernatant for oxidative stress indices.

Sections for histology were immediately fixed in 10% formol-saline prior to processing. The colon homogenate of each rat was prepared by grinding 0.5 g in ice-cold mortar with acid-washed sand in 5 ml of normal saline. Each homogenate was centrifuged at 3500 rpm for 5 minutes and the resultant supernatant was separated and left frozen at -20°C until required.

Biochemical assays

Superoxide dismutase activity was assayed by the method of [28] which involved the following of the auto-oxidation of adrenaline to adrenochrome at 420 nm. Catalase activity was assayed by using the method of [29], in which the decomposition of hydrogen peroxide was monitored at 480 nm. Glutathione peroxidase activity was determined by measuring the production of purpurogallen from pyrogallol at 420 nm [30].

Total protein was estimated using that involves the formation of biuride, commonly called “Biuret” method as described in Randox test kit assay leaflet. Malondialdehyde levels were measured in a colorimetric reaction with thiobarbituric acid as described by Buege and Aust.

Histology

Colon sections fixed in formol-saline were processed for light microscopy examination at the Department of Morid Anatomy and Histopathology, University of Benin Teaching Hospital (UBTH). The slides were examined and interpreted by a Consultant Pathologist.

Statistics

The results from biochemical assays are expressed as mean ± SEM. In order to establish whether the mean values were statistically significantly different from each other, Analysis of Variance (ANOVA) was done using SPSS software.

In order to know which means have differences that are significantly different, LSD multiple range tests was done by employing SPSS computer software. Values were considered significantly different at p ≤ 0.05.

The yield from ethanol extraction of Cycas circinalis leaf powder was 103.07/g residue.

Biochemical findings

The effects of the consumption of C. circinalis powder tainted feed and administered ethanolic extract of the powder on colon total protein, SOD, catalase and gluthatione peroxidase as well as MDA levels of the rats in various groups as shown in Table 1. Group B rats (cyacas tainted diet) had significantly (p ≤ 0.05) higher MDA level than any other group. However the MDA level of all the treatment groups increased significantly (p ≤ 0.05) compared to the control group. There was also a significant (p ≤ 0.05) decrease in the colon total protein level in all the treatment groups when compared to the control. The enzymes superoxide dismutase, catalase and glathatione peroxidase also decreased significantly (p ≤ 0.05) relative to the group.

Oral LD50 of ethanolic leaf extract of Cycas circinalis

No death was recorded at any of the doses administered. Oral LD50 was therefore determined to be over 5000 mg/kg body weight.

Histopathological observations

Stained section of control rat colon, revealed prominent lamina propria lined by distinct columnar epithelium and crypt of lieberkuhn (long arrow) bound by muscularis mucosa (short arrow), Figure 1.

Figure 1: Photomicrograph of section of colon from control rat (H and E, x100).

Section of colon from rat exposed to cycas tainted feed showed shorter and narrower lamina propria (Figure 2), (Long arrow) surrounded by inflammatory exudates. Columnar epithelium was deeply stained with marked nuclear atypia, reduced goblet cells with nuclear polarization.

Figure 2: Photomicrograph of section of colon from rat fed C. circinalis leaf powder tainted diet. (H and E, x100).

Section of rat exposed to 10 mg extract/kg body weight revealed the presence of increased of goblet cells indicated by long arrow (Figure 3).

Figure 3: Photomicrograph of section of colon from rat exposed to 10 mg extract per kg body weight (H and E, x100).

Besides other normal histological features of the colon epithelium, colon section for rat exposed to 30 mg extract per kg body weight, revealed the presence of pyknotic nucleus (Figure 4).

Figure 4: Photomicrograph of section of colon from rat exposed to 30 mg extract per kg body weight (H and E, x100).

Rat from the group exposed to 50 mg extract per kg body weight had colon with densely stained pyknotic nuclei. Also evident was nuclear stratification and mild fibrosis (long arrow), (Figure 5). Rat exposed to 80 mg extract per kg body weight had colon section revealed columnar epithelium that is deeply stained with marked nuclear atypia (Figure 6).

Figure 5: Photomicrograph of section of colon from rat exposed to 50 mg extract per kg body weight (H and E, x100).

Figure 6: Photomicrograph of section of colon from rat exposed to 80 mg extract per kg body weight (H and E, x100).

Section of colon from rat exposed to 100 mg extract per kg body weight is presented in Figure 7 . Columnar epithelium and crypt of lieberkuhn are clearly evident and have pyknotic nuclei that is densely

Figure 7: Photomicrograph of section of colon from rat exposed to 100 mg extract per kg body weight (H and E, x100).

Cycas, the only currently known genus of the family cycadaceae, are considered as fossil plants though they have evolved only about 12 million years ago [31]. The most studied cycas are Cycas revoluta and Cycas circinalis, which contains the carcinogenic toxin cycasin [9,11]. Cycasin is metabolized by plant, bacterial and mammalian β-glucosidases to liberate the aglycone, methylazoxylmethanol (MAM), which is the active metabolite of cycasin [23]. Since researchers became aware of the potent carcinogenic properties of cycasin and MAM, these agents have been employed as reliable animal models for inducing a wide range of intestinal cancers, including colon cancer [9].In the present study, the abnormal changes seen in the colon ultra-structure of rats given cycas-tainted diet (5% w/w) as well as 80 and 100 mg ethanol extract of the leaf per kg body weight which include inflammation, reduced number of goblet cells with nuclear polarization and nuclear atypia, are some of the early events in the onset of colorectal cancer [5,25,32]. Results from this study also show a significant increase in lipid peroxidation level evidenced by high MDA levels in the colon homogenate supernatant of rats fed cycas-tainted diet and those administered ethanolic extract of Cycas circinalis leaf. The most deleterious impact of oxidative stress is lipid peroxidation, which has been implicated in the pathogenesis of numerous diseases including cancer [33]. Lipid peroxidation changes the fluidity of cell membranes, reduces capacity to maintain and equilibrated concentration gradients, and increases membrane permeability and inflammation [34].

There was also depletion in colon total protein in the test groups as compared to the control group. The cycas-induced decrease in colon tissue protein is in agreement with colon tissue protein loss usually associated with most cancers [25]. The protein loss has been attributed to increased protein degradation and/or decreased protein synthesis [35, 36]. Proteins are also susceptible to ROS induced damage and are frequent target of increased production of free radicals [37]. ROS oxidizes structural proteins and inhibits proteolytic system. Such reactions leads to alteration of structural proteins or alteration of enzyme functions, which can result to a wide range of downstream functional consequences, such as inhibition of enzymatic and binding activities, increased or decreased uptake by cells, inactivation of DNA repair enzymes, and loss of fidelity of damaged DNA polymerases in replicating DNA [38].

Antioxidant enzymes, catalase, superoxide dismutase and glutathione peroxidase activities also decreased significantly in the rats fed cycas-tainted diet and the ethanolic leaf extract. There is an overwhelming evidence to indicate that oxidative stress, defined as an imbalance between oxidants and antioxidants in favour of the former, leads to many biochemical changes. The depletion of the antioxidant enzymes could be attributed to the primary defense system by these enzymes against the ROS generated by the increased lipid peroxidation. It is well known that SOD, catalase and GPx play important role as protective defense enzymes against lipid peroxidation in tissues [39]. GPx, an oxidative stress inducible enzyme plays a role in the peroxyl scavenging mechanism and in maintaining functional integration of cell membranes [40].The decreased catalase activities could be attributed to the fact that catalase is used by cells to defend against the toxic effect of hydrogen peroxide, which is generated by various reactions and/or environmental agents and by the action of superoxide dismutase enzymes while detoxifying superoxide anions [41]. The general decrease in the antioxidant defense system could be attributed to depletion of the enzymes in attempt to reduce the impact of cycasin-induced oxidative stress on the colorectal cells. Decreased antioxidant enzymes and increased oxidative stress suggest a strong relationship between oxidative stress and colorectal cancer [42]."

The results obtained in this study suggests evidence that Cyacs circinalis powder (5% w/w) and higher doses of the ethanolic leaf extract inducedss oxidative stress in the colon of rats and some ultrastructural changes in the colon which are indicators of early events in the development of colorectal cancer.