5-Fluorouracil And Curcumin With Pectin Coating As a Treatment Regimen For Titanium Dioxide Induced Colon Cancer Model


 Objective: Induction of colorectal cancer in Wister rats using titanium dioxide and dimethylhydrazine and treatment using the physical conjugate of 5-Fluorouracil and Curcumin, with a synergistic approach.Methods: Compatibility studies are evaluated by using FT-IR,Vero cell lines, and HCT-116 cell lines are used for evaluating the synergistic approach. This followed by induction using titanium dioxide and dimethylhydrazine in Wister rats and treatment using 5-Fluorouracil and Curcumin with pectin coating. Result: The samples were found to be compatible. The synergistic effect was obtained at 1:1, 1:2, 1:4, and 2:1 ratio, where 1:4 ratio shows a CI50 value of 0.896, selected further for the animal studies when studied in HCT 116 cell lines and found to be safe with Vero cell lines. Colorectal cancer was shown to be induced within 70 days of administration of titanium dioxide and dimethylhydrazine.1:4 ratio of 5-Fluorouracil and Curcumin (50:200) shows effective for the treatment of colorectal cancer within 28 days, proven using histopathology report, bodyweight analysis, and hematological reports.Conclusion: 5-Fluorouracil and curcumin (1:4) ratio works with a synergistic approach and was proven effective for the treatment of colorectal cancer induced bythe titanium dioxide and dimethylhydrazine.


Introduction
According to the International Agency for Cancer Research (IARC), colorectal cancer is the third most common cancer type worldwide during2020, with nearly 2 million new cases [Cao et al. 2021]. In 2013, colorectal cancer killed nearly one million people worldwide, making it the second leading cause of cancer-related death. Although the cause is unknown, changes in eating habits can prevent 70% of sporadic cases of colorectal cancer. Recently studies reported that there is a correlation between foodgrade titanium dioxide (TiO 2 ) with colorectal cancer. Because nanoparticles (NPs) such as food-gradeTiO 2 have been shown in experimental models to have negative effects, there has been an increase in concern about the carcinogenic activity on human consumption of nanoparticle-containing foods. TiO 2 , a whitening agent also known as E171, can enter the human digestive system and bloodstream when consumed orally, according to new research [Bischoff et al. 2021].
In one study, human volunteers were given TiO 2 at a dose of 5 mg/kg. E171 has a daily intake of 1 mg/kg body weight for adults and 2 mg/kg body weight for children due to its widespread use in candies [Losso 2021]. Furthermore, the average adult in the UK consumes 5.4 mg of TiO 2 per day. As a result, numerous studies have been conducted to investigate the effects of taking TiO 2 orally. After a ve-day dose of 8.6 mg/kg body weight, TiO 2 was found to reach the spleen, liver, and mesenteric lymph nodes, but tissue absorption was very low [Cao et al. 2020]. Rats with the adrenal gland developed necrotic cells in the cortex and pycnotic nuclei after being given 2 mg/kg body weight orally for ve days. The amount of black pigment in both healthy and ulcerative colitis children's Peyer's patches increased with age, and it was discovered in both cases to be food-grade TiO 2 . Similar deposits, which the researchers believe to be food-grade TiO 2 , were discovered in colon cancer, Crohn's disease, and Crohn's colitis without the disease [Cvetković et al. 2020, Brand et al. 2020]. TiO 2 was found in lymphoid tissue of patients with Crohn's disease, ulcerative colitis, and colonic carcinoma. In our research work we induced colorectal cancer in Wistar rats using TiO 2 -E171(with water) along with 1,2 Dimethyl Hydrazine (DMH) [Dudhipala et al. 2018] followed by its treatment using 5-Fluorouracil (5-FU), curcumin (CUR), and a combination of 5 colorectal cancer, more than 80% of it is catabolized by hepatic dihydropyrimidine dehydrogenase (DPD), while the remainder causes cell death by inhibiting RNA and DNA syntheses via uorodeoxyuridine monophosphate (FdUMP) and uouridine triphosphate (FdUMP) (FUTP). The most serious side effects of 5-FU are myelosuppression, diarrhea, cardiotoxicity, dermatitis, and mucositisand 5-FU is also possessing multidrug resistance (MDR). Hence, for overcoming this resistance part,the use of a natural chemosensitizer and chemotherapeutic agent-Curcumin is used. The combination of 5-FU and Curcumin can produce a synergistic effect and hence can overcome the MDR issues also. The use of this combination approach can reduce the dose of 5-FU and also the toxic effects. Since the absorption of curcumin takes place in the colorectal region and the safety pro le of curcumin even at a high dose, this combination approach was taken for further studies.
Gingival mucositis developed in approximately 80% of cancer patients who received 5-FU treatment [Bachmeier et al. 2019]. Anticancer chemotherapeutics that targets the small intestine have been linked to gingival mucositis. Mucositis is a painful condition that can last months or years and has a negative impact on cancer patients' quality of life [Crowder et al. 2018]. It also increases their chances of developing hematemesis, which can lead to neutropenia and malnutrition. As a result of this link, mucositis is now recognized as a clinically signi cant disease. It also makes effective chemotherapy and radiotherapy impossible, putting a stumbling block in the ght against cancer. The anti-in ammatory drug 5-FU shortens gut villi, deepens the crypt, raises the apoptosis index, increases myeloperoxidase (MPO) activity, and lowers glutathione (GSH) levels in rats [Fonseca et al. 2021].
Cancer drugs cause apoptosis in both cancerous and healthy cells. Apoptosis is caused by cancer drugs in healthy intestinal cells, which can lead to complications such as mucositis [Fonseca et al. 2021]. The small intestine's mucosal replacement cycle is only 3-4 days long, making it especially vulnerable to anticancer drugs. GI mucositis can have a negative long-term prognosis because it increases the need for intravenous nutrition, decreases nutritional intake, and increases the risk of infection throughout the body.
In Rat, this compound has been shown to inhibit skin, stomach, colon, and liver cancers. Curcumin, according to Goel et al., does not inhibit the expression of COX-1 mRNA or protein. Curcumin has been shown in humans to be a safe and effective colorectal cancer chemopreventive agent [Fonseca et al. 2020]. It has already been used in preclinical trials as a chemopreventive treatment, and the fact that these were successful suggests that curcumin's use will increase in the future.5-FU and CUR was coated with pectin for the speci c delivery of the drug to the colorectal region since the pectin act as a pH sensitive polymer and helps in targeted drug release [Du et al. 2020]. With the coating using pectin the codelivery can be possible through oral route which is more convenient than injections. Because of its widespread use and numerous bene ts as a chemopreventive agent for colon cancer, we hypothesize that 5-FU in combination with Curcumin can be effective for the treatment of colorectal cancer induced by chemical agents such as TiO 2 and DMH.

Materials used
TiO 2 was obtained as a gift sample from Bimal Pharma PVT, LTD Mumbai, DMH was purchased from Sigma-Aldrich, PVT, LTD, 5-FU and pectin was obtained from Avra synthesis PVT, LTD, Hyderabad, and Curcumin obtained as a gift sample from Himalaya PVT, LTD, Bangalore. All the samples were undergone with purity and con rmation before the initiation of the experiment.

Compatibility studies using FT-IR
During the combination, the compatibility of the ingredients used in the formulation is tested at the molecular level. Researchers used Fourier-transform infrared spectroscopy to learn more about molecular and chemical compatibility (FT-IR).
FT-IR (FT-IR Spectrometer, Shimadzu 8400-S, Japan) and potassium bromide (KBr) pellet method were used to investigate the interaction of 5-FU and CUR. In a 1:4 ratio, a physical mixture of 5-FU and CUR (1:1) was mixed with anhydrous KBr. The translucent pellet was created by compressing 100 mg of the nely powdered mixture using a hydraulic press at 15 tons of pressure. Individual pellets were scanned at a rate of 4 mm/s with a resolution of 2 cm. Any peak's presence or absence in the physical mixture was compared to the presence or absence of any peak in the pure drugs [Fonseca et

In Vitro studies-synergistic activity in HCT 116 cell lines
The MTT assay was used to assess the cell toxicity of various formulations in Vero and HCT 116 cells.
Cells were seeded at a density of 1x10 4 cells per well in 96-well culture plates and incubated for 24 hours.
The half-maximum inhibitory concentration (IC 50 ) ofsamples was determined. To investigate the synergy between 5-FU and curcumin in the combination system, the combination index (CI) was calculated. The following equation 1 was used to calculate CI: where CI 50 is the combination index calculated using the IC 50

Animals used
For this study, the JSS College of Pharmacy, Ooty, India provided 32 white 6-week-old Wister male rats.
After one week of acclimation, the rat was divided into four groups of six rats each. All the procedures are followed by using the IAEC guidelines (JSSCP/OT/IAEC/36/2018-2019).

Preparation of E171
Solution of Tio 2 nanoparticles dispersion was prepared by ultra-soni cation (Bandelin RK 100 H, Germany) method for 30 min in 1ml of distilled water (pH 7.0). The stock solution is prepared to contain a 1.0 mg/ml concentration. Beforethe administration of the injection, the solution was sonicated for 15 min once again for removing the agglomeration. The solution was further carried out for the zeta potential and particle size analysis [

Particle size and Zeta potential
The mean particle/globule size and zeta potential (ZP) of TiO 2 nanoparticles were measured using a zetasizer ZS 90. (Malvern Instruments, UK). A photon correlation spectroscopy technique was used to determine the mean particle/globule size, which analyses uctuations in dynamic light scattering caused by Brownian particle motion. The mean diameter of 10 mm diameter cells was measured at a 90° angle at 25°C. Because it re ects the electric charge on the particle surface, the ZP is an excellent tool for determining colloidal system physical stability. At 25°C, all size and ZP measurements were taken with disposable polystyrene cells and disposable plain folded capillary zeta cells diluted in the original

Induction of colorectal cancer in animals
The Wister rats were administered with 5mg/kg body weight of TiO 2 for 5 days per week and 1mg/kg bodyweight of DMH 1 day per week for 70 days, bodyweight, hematological parameters, and histopathology reports were considered [Litschauer et al. 2020]. The bodyweight of the animals was analyzed on weekly basis. The pictorial representation of the induction procedure is given in gure 1.

Histopathology report
Before being processed for histopathology, each colon tissue sample was immersed in a 10% formaldehyde solution. The tissues were dipped in molten liquid para n before being solidi ed into blocks, making slicing and staining much easier. Tissue-para n blocks were cut into 6 m thick sections using a rotary microtome (Leica, UK; Model No. RM2135). Tissue slices were stained with hematoxylin and eosin after being mounted on staining stands. Pathological examination of tissue slices was carried out using a digital microscope [Cisne et al. 2018].

Treatment
After the completion of 70 days, the treatment was started. Group one is taken as a control and administered with normal saline [Wolf et al. 2018]. In the second group,5-FU with pectin coatingwas administered, the third group with CUR with pectin coating and the fourth group with the combination of 5-FU and CUR with pectin coating. The procedure for the treatment group is given in gure 2.

Hematological Parameters
All the animals were con ned to blood collection at the time of sacri ce to measure hematological parameters. Blood was collected via a retro-orbital puncture. The above parameters were investigated to gain a better understanding of the curcumin and the 5-FU combination effect on speci c blood components.

Statistical analysis
Graphpad prism 9 software was used for evaluating the statistical signi cance. An unpaired two-tailed students t-test was used for determining the comparison of the two groups statistically, followed by oneway ANOVA with a Bonferroni correction for the multiple comparisons followed with a bartlett's test. All the experiments were conducted at least three times (n>3) (cell lines 3 times and for animal studies n-6) and expressed as mean ± standard deviation (±SD). The signi cance level was taken to be 95% (p<0.05).

Compatibility studies and purity studies using FT-IR
The 5-FU compatibility with CUR was tested using FTIR at the molecular and chemical levels, and the results are shown in gure 3 and Table 1   in Tables 2 (a) and 2 (b). The cytotoxic effect of the drugs and their combination was found to be safe when given to the normal cell lines. At a concentration of more than 48µg/ml, only the cytotoxic effect was found in the normal cells. Hence, the dose of the drug for producing synergistic activity was found to be safe for the normal cells. All the reports were taken in triplicate n=3

Particle size and Zeta potential
The TiO 2 size distribution showed a maximum peak of 269.7 nm, and a Z potential of-0.0218 V when titration at pH 7 distilled water. The transmittance was found to be 79.8%. The particle size and zeta potential results are given in gures 6 & 7 respectively.

Bodyweight analysis
From the beginning of the experiment till the sacri ce of the animals, the bodyweight of Wister rats was taken and recorded. Bodyweight was monitored constantly once a week on every seventh dayfrom the induction of TiO 2 and DMH for 10 weeks (70 days). Moreover, the initial and nal weights were measured, noted, and compared. The bodyweight of the rats was found to be changed from day 14 due to the induction of colorectal cancer followed by a reduction in the bodyweight, with the start of the treatment.
The bodyweight of group 4 was found to reach the normal weight within 28 days, as compared to groups 1, 2, and 3 at the end of the study. The results show that when 5-FU and CUR combination strategy is followed, there show a reduction in weight and coming to the normal weight as compared to when 5-FU and CUR (with pectin coating) are given alone. The results are given in gure 8 and Table 3. The data is represented in the mean ±SD (n=6 per group). The signi cance value-p-value was found to be p=0.0124, (p<0.05) R 2 Value was found to be 0.6233. (one-way ANOVA with a Bonferroni correction for multiple groups and two-tailed Student's t-test for individual groups).

Histopathology report
The colon tissues were subjected to H&E staining for evaluating the tissue characteristics of each group after the induction and treatment. The histopathology reports of group 1, group 2, group 3, and group 4 are given in gure 9 (a, b, c, d). The reports depict that there shows a change in the tissue pattern of the colon cells when TiO 2 and DMH were administered for the induction of cancer. The cancer growth was observed where after the treatment with 5-FU and CUR combination, the architecture of the cells was returned to be normal.

Hematological Parameters
Hematological parameters were performed and noted to evaluate the changes in the concentration in blood content before and after the administration of the CUR, 5-FU, and its combination in the rats. The results are given in Table 4

Discussion
Colorectal cancer is a major concern for people all over the world, regardless of socio-economic status.
Despite having lower mortality rates than developed-country populations, this gure is expected to catch Histopathology reports show that, in group 1, cancer growth is observed which was substantially reduced when treated by using a combination strategy. The weight analysis also shows with the same report. The weight of the animals comes to be normal when treated with 5-FU and CUR combination. Hematological parameter's analysis also depicts that the combination works better when compared to the drugs used alone, where the blood contents were found to be normal value.In conclusion, the combination of 5-FU and CUR in the ratio of 1:1, 1:2, 1:4, and 2:1 produce a synergistic effect in HCT-116 cell lines. Among which, the 1:4 ratio was further carried out for the in vivo studies by inducing colorectal cancer using chemicals TiO 2 and DMH.

Conclusion
Food-grade titanium dioxide was proven as the cause of colorectal cancer in human beings. This chemical moiety is present in almost all coloured foodstuffs (like candies). Hence, the risk of colorectal cancer in humans is increasing day by day. The laboratory animals such as rats were administered with titanium dioxide and dimethyl hydrazine and found with the colorectal cancer induction within 70 days. Then, the treatment of these animals was carried out by using a physical conjugation of 5-FU and CUR.
Which was found to be effective when taken in a 1:4 ratio. They produce a synergistic property and are found as an effective treatment regimen in Wister rats which was proven using weight variation, histopathology report, and also hematological parameter's evaluation.     Zeta potential report.

Figure 8
Bodyweight analysis report weekly. The data is represented in the mean ±SD (n=6 per group). The signi cance value-p-value was found to be p=0.0124, (p<0.05) R2 Value was found to be 0.6233. (oneway ANOVA with a Bonferroni correction for multiple groups and two-tailed Student's t-test for individual groups).  Haematological parameters test report. The data is represented in the mean ±SD (n=6 per group). The signi cance value-p-value was found to be p<0.0001, (statistically signi cant) R2 Value was found to be 0.8851. (one-way ANOVA with a Bonferroni correction for multiple groups and two-tailed Student's t-test for individual groups). Bartlett's statistic correlation was found to be 6.154 with a P-value of 0.1044, there is no SD's signi cant difference (P<0.05) found.