Efficacies Of Chlorophyllin And Chlorophyll

Published Date: 02 Nov 2017

For the chlorophyllin and chlorophyll used, the samples were tested at 2000p.p.m for consistent result between chlorophyllin and chlorophyll. The properly established and designed positive control gives a tremendous reduction from 57% incidence for DBC alone to 12% for chemoprevention when using 2000 p.p.m CHL, whereas for 2000p.p.m of purified chlorophyll almost same reduction from 57% to 14% observed. (McQuistan et al., 2012). Finally for 2000p.p.m of extracted chlorophyll the results give a reduced liver tumor incidence from 57% to 31% (McQuistan et al., 2012). From the result the tumor incidence reduces as chlorophyllin is used and it showed a better result compared to extracted chlorophyll and purified chlorophyll (McQuistan et al., 2012). Even though purified chlorophyll gives a comparative and almost same the result as chlorophyllin the process of preparing purified chlorophyll is very complicated and tedious. It is proven from this that the same concentration of chlorophyllin given to the trouts provides more chemopreventive activities compared to chlorophyll (McQuistan et al., 2012). Hence chlorophyllin provides a good alternative having chemoprevention properties compared to chlorophyll (McQuistan et al., 2012).

2.1 Effects of chlorophyllin to reduce DNA adduct of mutagenic polycyclic aromatic hydrocarbon (PAHs)

In this section effect of chlorophyllin on two polycyclic aromatic hydrocarbons will be reviewed. Both case studies might look the same but different approaches were used to determine the effectiveness of chlorophyllin on the PAHs. The first case study emphasizes on the dose of chlorophyllin needed and dose of carcinogen needed to effectively reduce the tumour formation or DNA adducts formation involves in shasha rainbow trouts. Another approach is the use of transgenic mice lack the protective mechanism towards DNA repairs which then able to determine whether chlorophyllin is able to confer protective mechanism even in insufficient protective mechanism condition on the animal itself.

Case Study 1

Here is a review of an expanded analysis of CHL chemoprevention using the potent environmental hydrocarbon dibenzo[a,l]pyrene (DBP) a type of polycyclic aromatic hydrocarbons (PAHs) conducted by Pratt et al. in 2007. A dose–dose matrix was designed and employed approximate over 12 000 rainbow trout to evaluate the complex and complicated relationships among dietary carcinogen dose, anti-carcinogen dose (chlorophyllin), carcinogen–DNA adduct levels at exposure and eventual tumor outcome in two target organs, the liver and stomach of rainbow trout (Pratt et al., 2007). Chlorophyllin (CHL) has been proven to be effective against environmental PAHs. According to Goldman, R in 2003 human dietary exposure to PAHs has been approximated at 3 mg/day for non-smokers, an exposure comparable to the estimated 2–5 mg PAH/packs of cigarettes. It was previously proven and demonstrated that dietary DBP co-treatment with CHL eventually reduced tumour response in multiple target organs in rainbow trout (Reddy et al., 1999). Previous studies show that 500 p.p.m. DBP exceeds a maximum dose which is tolerable, and that 200p.p.m. is sufficient for sufficient tumor response (Pratt et al., 2007)

In this study, shasha rainbow trouts were used. A total 12 350 trout were selected and divided, into 130 fish per tank into 95 tanks in randomized order. The concentrations of DBP used in this study were chosen as log (10.1, 28.4, 80.0 and 225 p.p.m.). In this study, the CHL was pre-fed 1 week at different concentration of 0, 1500, 3000, 4500 or 6000 p.p.m. , then co-fed with the different amount doses of DBP for 4 weeks, followed by 1 week of post-initiation feeding with CHL, to assure the presence of CHL throughout DBP metabolism. A higher dosage of DBP was used for groups receiving 4500 and 6000 p.p.m. CHL in order to compensate for the anticipated substantial prevention of tumor response. Twenty nine days after starting with carcinogen feeding, five fish were removed from each tank and euthanized following by DNA purification from thawed and pooled liver samples. The purified DNAs were post-labeled for futher process . The remaining fish in each tank were fed OTD (OREGON TEST DIET ) and allowed to grow for additional 10 months,for tumor incidence evaluation and measurement followed by evaluation of the tumor produced (Pratt et al., 2007).

Table.2.1.1 The result of tumor incidence, liver tumor index and tumor type where the rainbow trout were pre-fed with CHL for 1 week at 0,1500,3000,4500 and 6000p.p.m then co-fed with varying doses of DBP for four weeks. (Pratt et al., 2007)

Table 2.1.1 shows there was no evidence of DBP or CHL dosages affecting the mean body weight of trouts. This table also shows that tumor incidence of liver, stomach and swim bladder of trouts observed at the 10th month after the treatment of DBP dosage ceased. (Pratt et al., 2007) Tumor incidence was the most in liver followed by the stomach and swim bladder at each DBP dose, followed. From the table we can see that as at 0 concentration of DBP there is no tumor incidence in all liver, stomach and swim bladder of the trouts. This shows that DBP is indeed a carcinogen that can initiate tumor incident in trouts and possibly human. The tumor incidence is highest when a 225p.p.m DBP dose is given alone without CHL for all liver, stomach and swim bladder. (Pratt et al., 2007) When the trouts were fed with increasing amount of CHL before commencement of 225p.p.m DBP, the tumor incidence in liver, stomach and swim bladder dropped tremendously and for swim bladder the tumor incidence is close to 0, with 0.36 when the touts were fed with 4500p.p.m of CHL. Hence it can be sad that for a consistent amount of DBP used, increasing CHL dosage reduces tumor incidence in liver, stomach and swim bladder of the trouts. The decrement of tumor might not be consistent as we can see for swim bladder at 6000p.p.m of CHL and 225p.p.m of DBP fed with 0.67 tumor incidences, which are higher than 4500p.p.m of CHL used with only 0.36 tumor incidences. (Pratt et al., 2007) From this table it can be conclude that increment of CHL doses at same DBP concentration (up to 225p.p.m) decreases tumor incidence in liver and stomach but not swim bladder of the trouts.

Table 2.1.2 Result of DNA adduct found in liver and stomach of rainbow trout after given different doses of DBP and CHL. (Pratt et al., 2007)

Table 2.1.2 shows the result on DNA adducts formation after 4 weeks of different concentration of DBP and CHL feeding commence. The table shows the total DNA adduct formation in both liver and stomach of the trout, the table also record down the amount of polar and non-polar DNA adducts in liver and stomach of the trouts. Where the 4 weeks of dietary DBP treatment was guaranteed will produce a stable DNA adducts formation in liver and stomach of the trouts. (Pratt et al., 2007) From the table we can see that when no DBP were given to the trouts there were adducts formation both in liver and stomach of the trouts. As the concentration of DBP increases the adduct formation increases. Trouts fed with DBP concentration of 225p.p.m and 0p.p.m concentration of CHL give the highest adducts formation in liver and stomach of trouts. (Pratt et al., 2007) It can be seen from the table that for trouts fed with 225p.p.m of DBP doses, the adducts formation in liver decreases as concentration of CHL increases from 1500p.p.m ,3000p.p.m, and 4500p.p.m where there is a slight increase of DNA adducts formation when the trouts were treated with 6000p.p.m of CHL. Whereas, for DNA adducts formation in stomach for 225p.p.m of DBP dosage, increasing amount of CHL from 1500p.p.m, 3000p.p.m, 4500p.p.m and 6000p.p.m showed a decrease trend of adducts formation. (Pratt et al., 2007) From this table it can be concluded that trouts given 225p.p.m DBP and increasing amount of CHL in their dietary doses reduces the adduct formation both in liver and stomach.

Figure 2.1.1 Chromatogram of in vivo DBP-DNA adducts from shasha rainbow trouts after 33P post-labelling and HPLC analysis. (Pratt et al., 2007)

As identification of every DBP–DNA adduct in liver and stomach was beyond the scope of this study post-labeling/HPLC protocol (Harttig and Bailey, 1998) is used to resolve and identify major and minor adduct peaks which them will be able to examine the effects of DBP and/or CHL dose on adduct profile as shown in Figure 2.1.1 .As we can see from Figure 2.1.1 the counts per minute of 33P reduces in B and D where B is the liver adducts and D is the stomach adducts of trouts fed with 225p.p.m DBP and 1500p.p.m chlorophyllin. While A is the liver profile and C is the stomach adducts trouts profile of 33P chromatogram fed with 250 p.p.m DBP alone The liver DBP– DNA adduct profile A eluting within 10-20min were mostly polar adduct peaks eluting and several smaller non-polar peaks eluting in the 35–65 min range. (Pratt et al., 2007) Increasing concentrations of CHL at each doses of DBP resulted in a comparatively uniform decrement in the DNA-adduct profile. (Pratt et al., 2007) This is illustrated in B, which shows the magnitude and uniform nature of the decrease in DBP adduct profile at 1500 p.p.m. CHL. (Pratt et al., 2007) Dose dependent formation of tumor in the liver, stomach and swim bladder observed after 10months the administration and feeding of DBP ceased (Table 2.1) (Pratt et al., 2007). Tumor incidence was greatest in liver at each DBP dose, followed by the stomach and then swim bladder which can be seen from Table 2.1.1. Addition of CHL to the diets generally resulted in a dose-dependent reduction in tumor formation across all doses of DBP. (Pratt et al., 2007) Evident result was shown that rainbow trout treated with CHL reduces in DNA adduct in both liver and stomach.

In summary, the present study used 12 350 shasha rainbow trouts to successfully elucidate the complex interrelationships between carcinogen dose applied, anti-carcinogen dose applied, target organ DNA adduct biomarkers (effective dose received), and eventual tumor outcome. By molecular dose analysis, CHL-mediated alterations in DBP–DNA adduct levels were generally predictive of eventual reduction in hepatic tumor incidence and multiplicity, but only below 80 p.p.m. DBP (Pratt et al., 2007). According to the result at higher DBP dose, however, lower doses of chlorophyllin failed to reduce hepatic tumor incidence, but the highest chlorophyllin dose instead increased tumor division than reducing it, and DNA adducts failed to be predictive biomarkers of eventual tumor outcome (Pratt et al., 2007). These results showed that the importance of designing endpoint dose–response proportionality in selecting carcinogen doses for chemoprevention studies. (Pratt et al., 2007).

Case Study 2

In the following review Benzo[a]pyrene (BP) a PAH is used as carcinogen compound where DNA adduct formation in DNA repair–deficient p53 haploinsufficient [Xpa(−/−)p53(+/−)] and wild-type mice fed BP and BP plus chlorophyllin for 28 days is used to determined the effectiveness of chlorophyllin. BP is a widely distributed environmental carcinogen, , exposure to BP can occur through different type of diets, lifestyle, and occupations. After exposure, BP undergoes metabolism by Phase I enzymes, predominantly cytochrome P450s (CYPs), to variety of genotoxic metabolites which were able to undergo reaction with cellular metabolites. A major BP metabolite, r7,t8-dihydroxy-t- 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), binds deoxyguanosine in DNA to form, r7,t8,t9-trihydroxy-c-10-(N2-deoxyguanosyl)7,8,9,10tetrahydrobenzo[a]pyrene (BPdG). Below shows the conversion of benzo(a)pyrene a polycyclic aromatic hydrocarbon to BPdG, a type of DNA adducts.

Figure 2.1.3 The metabolic conversion of benzo(a)pyrene (BP) into DNA-adducts. (Weinstein, I. B. et al., 1976)

Benzo(a)pyrene goes through several steps as can be seen in Figure 2.1.3(a) as it is made more water soluble prior to excretion. One of the intermediates in this process, a diol epoxide Figure 2.1.3(3), is capable of reacting with guanine in DNA Figure 2.1.3(b). This reaction leads to a distortion of the DNA molecule Figure 2.1.3(c) and mutations.

The formation of BPdG adducts in important key tumor suppressor genes, oncogenes and gatekeeper genes, causing leading to mutations in animal models, has been considered to be important in starting the multi-step process of chemical carcinogenesis (John, et al., 2012). In response to carcinogen exposure in this case, BP biological organisms uses different kind of intrinsic protective mechanisms. The protective mechanism includes nucleotide excision repair (NER). NER is likely to commence for removal of DNA damaging UV-induced photo lesions. NER also actively removes large molecular weight DNA adducts such as the BPdG (John, et al., 2012). The inability to repair for the UV-induced lesions because of deficient in the nucleotide excision repair pathway results in the disease Xeroderma pigmentosum (XP). XP is an inherited autosomal recessive disorder characterized by excessive sensitivity of skin to UV radiation and accelerated skin cancer induction. There are 30–40 different polypeptides involved in the process of NER, and the XP complementation group A (Xpa) protein is thought to have a key role in binding to damaged DNA followed by the positioning the repair machinery around the lesion . However, Xpa-deficient mice which are lack almost all of the required NER and are more susceptible to enhanced cancer induction when exposed to various chemical carcinogens, including BP (John, et al., 2012). p53 is a multi-functional tumor suppressor protein, which play vital role in inhibiting the cause of cancer or tumorgenerisis and is usually mutated in cancer patients/individual. In part, p53 provides protection against tumor induction by delaying down the replication of damaged DNA. Hence to allow for damage removal by NER as the replication is slowed down. Therefore, transgenic mice which are deficient in both Xpa and p53 provide as an attractive and proper model for the study of genotoxic carcinogens such as BP. (John, et al., 2012). In comparison with their wild type counterparts, Xpa (−/−) p53 (+/−) mice have the characteristics to amplify the susceptibility to chemical carcinogen induced tumors by carcinogen such as BP in this studies. Hence the DNA adduct processing in esophagi, livers and lungs of WT and Xpa(−/−)p53(+/−) mice fed BP for 28 days is established, a time period known for the formation of stable state DNA adduct formation. CHL would reduce BP–DNA adduct formation in target organs, thus protecting them against tumor cell production or tumorgenesis. (John, et al., 2012).

In this study the method involved include the Xpa(−/−)p53(+/−) mice, which were generated on a C57BL/6 background (Hoogervorst et al., 2003). Following birth of the mouse offspring, mouse were paw tattooed and tail- clipped for 10 days which subsequently the DNA from the tail clips was used to genotype the mice, using primer sequences for Xpa and p53 (Hoogervorst et al., 2003). A pathogen free house is provided for the mouse with a climate –controlled facility and also a 12hours of light and dark cycle. Dietary exposure was started when the mice were at 6–8 weeks of age and carried out for a total of 4 weeks. The animal models were subjected to one of the following treatment regimes: powdered NIH 31 diet (control); powdered NIH 31 diet containing 100 p.p.m BP; powdered NIH 31 diet containing 100 p.p.m BP + 0.3% (3000 ppm) CHL or powdered NIH 31 diet containing 0.3% CHL alone. All mice were given food source and water ad libitum. Per treatment group, 4–10 animals were used with approximately equal numbers of males and females to make it equal.

Following 4 weeks of dietary exposure, mice were euthanized and tissues flash frozen and stored until DNA was extracted (John et al., 2012). Upon thawing, tissues were homogenized and incubated overnight at 37°C. The next day, DNA was extracted from the esophagi, lungs and livers using DNeasy Blood Maxi kits (Qiagen).Then it was followed by DNA adduct analyses where two approaches were used to evaluate the levels of DNA damage. Where the first approach, the BPdG adduct was analyzed by HPLC/ES-MS/MS, (Beland et al., 2005) where this method will assess BPdG adducts only. Where the second approach involved the CIA (Chemiluminescence Immunoassay) which is done by using a rabbit polyclonal antiserum elicited against BPDE-modified DNA (Divi et al., 2002). Even though the immunogen was modified DNA with BPdG, the antiserum still able to cross-reacts with the family of carcinogenic PAHs bound to DNA (Weston et al., 1989), and also all the stable state BP DNA adducts formed.

Figure.2.1.4: DNA adducts (per 108 nucleotides) induced in (A) esophagus, (B) liver and (C) lung of WT and Xpa(−/−)p53(+/−) male (○) and female (● ) mice fed 100 ppm BP in the diet for 28 days. (John et al., 2012).

From Figure 2.1.4 we can see the result of DNA adducts formation from two approaches used in measuring the adducts, the left side represent the DNA adducts (BPdG) formation measured by HPLC/ES-MS/MS and the right side of the figure show the DNA adducts (BP-DNA adducts) formation measured by BPDE-DNA CIA. From the figure it can also be seen DNA adducts formation in three different parts and organs of the mice A, B C, which are esophagus, liver and lung respectively. (John et al., 2012). The important message brought up by this figure is that wild type mice which having complete protective mechanism have a lower level of DNA adducts formation while the transgenic mice with insufficient protective mechanism towards BP have a higher level of adducts no matter the gender of the mice. (John, K., et al., 2012). From the y-axis of the figure it can be seen that DNA-adducts formation is the most in the liver followed by esophagus and lung of the mice. (John, K., et al., 2012). From the figure it can be seen that the lungs of wild type mice are very resistant to formation of BPdG adducts but the transgenic mice are very sensitive to BP and hence easily and high number of BPdG adducts form. It can be concluded that mice’s liver is most susceptible towards BP to form DNA adducts which include BP-DNA adducts and BPdG followed by esophagus and lung.

Table.2.1.3 Comparison for males and females combined, for BPdG adducts (per 108 nucleotides) determined by HPLC/ES-MS/MS in WT and Xpa(−/−)p53(+/−) mice fed BP or BP + CHL for 28 days. (John et al., 2012).

From this table important information can be seen where the wild type mice have a lower DNA adducts compared to the transgenic mice when they are fed with BP alone or given BP + CHL diet. This table also shows that the mice esophagus are most susceptible to BP DNA adducts formation with 34.4 +/-2.4 BPdG DNA adducts in per 108 nucleotides. (John et al., 2012). Other information that can be seen from this table is that administration of CHL at 3000p.p.m decreases the BPdG formation by 30.5 % in esophagus for wild type mice. However it was not observed the same decrement in BPdG adducts formation in liver and lungs of wild type mice, as there is an increase in BPdG adducts when the mice were administrated with 3000p.p.m of CHL. For transgenic mice there is decrement of BPdG adducts formation in esophagus, liver and lung when adduct were compared for BP diet alone or BP + CHL diet. This shows some inconsistence of the result as there are difference in the decrement of wild type and transgenic mice BPdG adducts formation.

Table 2.1.4 Comparison, for males and females combined, for BP–DNA adducts (per 108 nucleotides) determined by BPDE–DNA CIA in WT and Xpa(−/−)p53(+/−) mice fed BP or BP + CHL for 28 days. (John et al., 2012).

In Table 2.1.4 it was showed the measurement of total BP-DNA adducts measured with chemiluminissence assay. (John et al., 2012). For this measurement, it was not predicted that the BP-DNA adducts for wild type mice treated with 100 p.p.m of BP and 3000p.p.m of CHL to be much more compared to mice treated with BP only. Hence the wild type mice showed a reverse pattern of result as adducts should be reduced when the mice were given diet containing CHL. These result shows contradict expected result as CHL function is to reduce adducts formation. The increment of adducts is the most in wild type mice given BP + CHL diet compared to BP diet only. The data is considered to be consistent as the increment not only occurs to one organ of the mice but all esophagus, lung and liver of wild type mice also show since increment of BP-DNA adducts. The increment is as much as 409% for wild type mice liver which were treated with both BP and CHL compared to wild type mice treated with only BP. (John et al., 2012). While for transgenic mice, there is reduction in BP-DNA adducts for mice treated with both BP and CHL, but the result isn’t consistent as there is increment of DNA- adducts formation for the lung of the animal models, the adducts increase by 20.34%. The result is not consistent as the expected result should be decrement in adducts in mice treated with both BP and CHL but there is instead increment in some part of the mice let it be the wild type or transgenic mice.

It was expected that, the Xpa(−/−)p53(+/−) mice, which are deficient in NER and partially insufficient in p53 tumor suppressor protection, typically showed higher levels of DNA damage in esophagus, liver and lung, than their WT counterparts, when fed 100 p.p.m BP in the diet for 28 days. These results were consistent with data observed in several studies that administered PAHs to Xpa(−/−) or Xpa(−/−)p53(+/−) mice, in which the end points were DNA damage, mutagenesis or tumorigenesis. It was shown that CHL did confer protective against formation of DNA adducts but not in a very high percentage in mice and a repetitive study and research must be carried out for a more accurate result (John et al., 2012). Furthermore, different types of measuring adducts were employed in this study. Hence it could not be decided whether CHL do confer protection 100% towards formation of adducts in mice.

2.2 Effects of chlorophyllin on mutagenic aflatoxin

In this section effects on chlorophyllin on aflatoxins will be reviewed starting with the introduction of aflatoxin and how it is harmful and a carcinogen. Followed by case study reviewing the intervention of CHL with aflatoxin in human volunteers.

Aflatoxins, particularly aflatoxin B1 (AFB1), mycotoxins occurring naturally and found in contaminated food that is associated with the growth of two types of mold: Aspergillus flavus and Aspergillus parasiticus (Jubert et al., 2009) found in maize, peanuts, soy sauce and fermented soybeans is proven and considered to be highly carcinogenic in animal models, including fish, rodents, and nonhuman primates (Egner et al., 2003). Aflatoxin was classified as human carcinogen by the International Agency on Research in Cancer in 1993. Aflatoxin occurring in high level or concentration combined with hepatitis B virus has been considered to be able to act in a synergistic manner that will cause and increase the risk of getting hepatocellular carcinoma (HCC) a type of liver cancer which are the most common type of liver cancer (Jubert et al., 2009) . Epidemiological and experimental research also proves the strong link between aflatoxin exposure and hepatocellular carcinoma (HCC) even in patient without hepatitis B virus infection (Egner et al., 2003). HCC is one of the most dominant cancers worldwide with incidence rates highest in geographical regions of Africa and Asia with high heat, humidity climacteric condition and poor food storage conditions. (Egner et al., 2003). In the Peoples Republic of China, HCC accounts for 300,000 deaths annually and is the third leading cause of cancer mortality (Egner et al., 2003). Hence it can be said that aflatoxin causes cancer in liver most of the time. Aflatoxin can be categorized as hepatocarcinogen since it mostly cause liver cancer (Egner et al., 2003).

Figure 2.2.1: Pathway for formation of aflatoxin-N7-guanine after exposure to aflatoxin B1 (AFB1) (Egner et al., 2001)

The pathway shown above shows the production of aflatoxin-DNA adducts which will cause hepatocellular carcinoma in humans and animal model. This aflatoxin–DNA adduct excretion product is used in many studies by researches. This is because this adducts are able to serves as a biomarker of the biologically effective dose of aflatoxin and elevated levels which are associated with increased risk of liver cancer caused by hepatocellular carcinoma particularly. (Egner et al., 2001)

Case Study 3

The case study to be reviewed involves the effects of chlorophyllin on low dose aflatoxin B1 pharmacokinetics in human volunteers (Jubert et al., 2009). The purpose of the present study was to investigate AFB1 pharmacokinetics in human volunteers by using the micro-dosing techniques and accelerator mass spectrometry (AMS) analysis, and also to explore possible effects of Chla and CHL co treatment on AFB1 pharmacokinetic parameters (Jubert et al., 2009). The study involves the investigation of three volunteers who received a 30-ng dose of 14C-labeled aflatoxin, without or with CHL or Chla co treatment for investigation of kinetics of low-dose AFB1 (Jubert et al., 2009) Only the effects of CHL will be discussed. The mechanisms that produce this effect in humans are undefined but may include limiting absorption in the intestine, systemic complexation with bioavailable forms of CHL, or induction of phase II enzymes leading to increased aflatoxin metabolism (Egner et al., 2003).

In this studies the method involved is very complicates as this is a pharmacokinetics study. 14CAFB1 (radioactively labelled aflatoxin) was used in this study and the radiochemical and its chemical purity was confirmed to be 97% by high performance liquid chromatography. The high performance liquid chromatography was equipped with online radioisotope detector and photoiodide array detector. CHL was the same lot used in the China intervention trial for a comparable result obtained (Egner et al., 2001). Capsules of CHL were prepared by filling them with appropriate ingredients and each of the capsule was prepared fresh as needed to prevent any contamination and expired ingredients also to standardize the result as there are different volunteers. The protocol for this study and the designation were approved by both Institutional Review Boards at OSU and Lawrence Livermore National Laboratory (LLNL) before the study was conducted. Volunteers were recruited from a group where they all had the sufficient and proper scientific knowledge and were aware of the consequences of this study to evaluate for the toxicologic issues and risks. Volunteer characteristics were provided in Table 3.1. (Jubert et al., 2009)

Table 2.2.1: Characteristics of the 5 volunteers who participate in this study. (Jubert et al., 2009)

The design of this treatment were divided into three protocols, where the first protocols were where at 8 am after fasting each volunteers was given gelatin capsule containing 14CAFB1 which they were allowed to swallow with 100mL of water, normal eating and drinking were allowed and resumed 10 am onwards (Jubert et al., 2009). Protocol 2 was the same as protocol 1 the difference is just that the capsule contain extra 150 mg of purified chlorophyll (Jubert et al., 2009). Protocol 3 was also same as protocol 1 the differences is the capsule given having extra 150 mg of CHL (Jubert et al., 2009). All of the 3 protocols were repeated thrice for a consistent and reliable result. For each volunteers their blood sample of 3 mL were collected by qualified nurses at time 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3, 4, 8, 12, 24, 24, 48, 72 hours (Jubert et al., 2009). Each volunteers also required providing their urine sample at intervals of 0 to 2h, 2 to 4h, 4 to 8h, 8 to 12h, 12 to 24h, and every subsequent 24 hours until the study ended (Jubert et al., 2009). After the blood and urine collection process was done, AMS analysis of the plasma and urine sample was conducted to measure the ration of 14C to total carbon (Jubert et al., 2009).

Figure 2.2.2: Cumulative urinary excretion of aflatoxin from subject 1, 2, and 5 (three volunteers of this study). The aflatoxin amount is derived from AMS analysis based on total 14C. (Jubert et al., 2009).

From this figure it was shown the urine excreted from 3 subjects over the 72 hour period for aflatoxin challange with and without intervention of chlorophyll or CHL. From the result it can be seen that for the first 24 hours the excretion was rapid with high percentage of the total urine aflatoxin equivalents (Jubert et al., 2009).For the entire subject involved the urine aflatoxin excretion is the highest when the subjects were given with protocol 1 throughout the 72 hours period. There is effect when the subjects were given different protocol as they received intervention of chlorophyllin and chlorophyll. It can be seen from figure 3.2 throughout the 72 hours of treatment, there is a consistent style towards reduced urinary excretion, and the effect of CHL showed a significant reduced in urinary excretion; even though the reduction is not as significant as intervention by chlorophyll (Jubert et al., 2009).It was reported subject 1 excreted an average of 29.0 +/- 2.31% of on the dose in the 72 hours period when treated with protocol 1 whereas intervention with CHL reduces the excretion to an average of 24.0 +/- 2.95% (Jubert et al., 2009). Subject 2 also give the similar effects with an average of 34.4+/- 5.6% for receiving protocol 1 treatment and 24.1 +/-5.37% for intervention with CHL, whereas subject 5 excreted 33.3 +/- 2.79% of the aflatoxin dose and 21.2 +/- 2.17% with CHL (Jubert et al., 2009).Hence from this figure, it can be conclude that CHL do reduced the total urinary excretion compared to treatment with only aflatoxin.

Figure 2.2.3: Pharmacokinetics profile of aflatoxin equivalents from plasma samples collected at 0.25, 0.5, 0.45, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 8.0 hours from subject 1, 2, 3 and 5 (Jubert et al., 2009).

It was reported that from Figure 2.2.3 it can be seen that absorption of AFB1 is equivalents into systemic circulation which was rapid with peak concentration that were reached within 1 hour. Subject 3 only received protocol 1 and incomplete protocols 2 and 3, but the one intervention trial given with CHL produces a result consistent with protection by CHL. From all four graphs, the trend is obvious that at the first two hour the plasma aflatoxin (pg/mL) increases and gradually decreases from 2.0 hour onwards till end of 8.0 hour. The trend is obeyed by all individuals whether the individual receiving protocol 1, 2 or 3.

Table 2.2.2 Individual pharmacokinetics parameter of aflatoxin equivalents (Jubert et al., 2009).

This table shows the pharmacokinetics parameters of subject 1, 2 and 5 who received treatment of all three protocols designed. As it can be seen from the table, subject 1 shows a sturdy and highly significant reduction in Cmax(P < 0.01) for protocol 2 which given capsule of aflatoxin with chlorophyll. (Jubert et al., 2009). Subject 1 gives a less pronounced response toward CHL as compared to intervention by chlorophyll. In comparison subject 2 does not show the same in Cmax (P < 0.27) for protocol 2. (Jubert et al., 2009). Subject 5 also give a different respond as subject 5 shows to be responded to both chlorophyll and CHL intervention with a significant reduction in Cmax (P < 0.05) for both protocols. (Jubert et al., 2009).

In this study the usage of AMS technique allows for the investigation of the absorption and pharmacokinetics of AFB1 and its metabolism that was conducted on the volunteer subject. The importance of this study is to establish the kinetics of low-dose aflatoxins absorption in human volunteers. This is important as we can understand the consequences and the aflatoxins absorption in human and the effect of intervention by CHL.

Properties and mechanism of action of chlorophyllin that make it a potent anticancer activity

In this section it will be reviewed the action of chlorophyllin as antiapoptotic agent, also to review the action of chlorophyllin in inhibiting canonical NF-κB signaling pathway and induces intrinsic apoptosis. It is this characteristics and action of chlorophyllin that attribute to its chemopreventive, antimutagenic and anticarcinogenic properties action towards carcinogenesis.

2.3.1 Antiapoptotic and immunomodulatory effects of chlorophyllin

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) were generated by rapid uptake of oxygen by macrophages during the innate immune response. The function of ROS and RNS is to destroy the invading pathogens as ROS is capable of activation of human peripheral dendritic cells and also proliferation of lymphocytes. Even though generation of ROS is beneficial for destroying invading pathogens, it is also well known that generation of ROS brought about the ionizing radiation which damages the cell. Due to the ionizing damage that was brought up by ROS it results in immunosuppression of the organism, hence more susceptible to causes cancer. Chlorophyllin which are antioxidants may help in preventing radiation induced damage that is brought up by ROS by scavenging ROS. Hence these properties of chlorophyllin was study by Sharma et al., in 2007 where experiments were conducted to demonstrate the effect of CHL on radiation induced immunosupression and modulation of immune response in mice was examined. It was then thought that these properties of chlorophyllin attribute it for being a research for chemopreventive study as it prevents the immune response to be weakened.

In order to run this study eight to ten week old BALB/c strains of mice weight approximately 20-25g were reared. The study involved in vivo and in vitro gamma irradiation carried out using junior theratron telepathy machine. Mice were sacrificed 24hours after irradiation. As for the in vitro experiment CHL was added in culture medium at the time of commencement of the culture. For ex vivo or in vivo experiments CHL was dissolved in RPMI medium at different concentration of CHL and injected into the BALB/c mice. After the mice were sacrificed the spleen cells were obtained by squeezing the spleen through nylon mesh. In order to obtain only lymphocytes from the spleen the red blood cells were lysed by hypotonic solution leaving only lymphocytes. Lymphocytes were hen stained with CFSE for 5 minutes at 37 °C. Two million lymphocytes were stimulated with concanavalin A (Con A) and in the presence of CHL and were cultured. Untreated cells served as a control. The cells were then determined its percentage in different phases in cell cycle and the percentage of cell apoptosis were determined by flow cytometry. It was followed by the estimation of bcl-2 and bcl-xL mRNA expression by semiquantitative RT-PCR. (Sharma et al., 2007) Then the size of the spleen and cellularity was determined to see the effect of Con A and CHL. (Sharma et al., 2007)

Figure 2.3.1.1 Effect of CHL on Con A induced proliferation cell cycle and apoptosis: Effect of CHL on proliferative response of spleen cells to (a) Con A in vitro. CFSE labeled spleen cells were stimulated with the mitogen in presence of various concentration of CHL at 37°C in RPMI 1640 medium supplemented with 1-% FCS in a 95% air /5% CO2 atmosphere for 72 hours and 20,000 cells were acquired in FACS. Percent daughter cells were calculated from decrease in mean fluorescence intensity using Cellquest® software. Each bar represents percentage of daughter cells after more than four divisions. (b) Effect of CHL on cell cycle progression in Con A stimulated lymphocytes. One million lymphocytes were stimulated with Con A in presence of different concentrations of CHL and cells were stained with propidium iodide solution. Each bar represents percentage of cells containing more than 2n DNA (S/G2 + M phase). (Sharma et al., 2007)

Figure 2.3.1.1(a) shows the effect of CHL on Con A induced proliferation of spleen lymphocytes of BALB/c mice. CHL concentration up to 10µM did not give inhibition to Con A action of inducing proliferation of T cells. However at higher concentration of CHL at 50µM there is a tremendous reduced in number of daughter cells observed compared to when the CHL concentration was lower. When the concentration of CHL was increased to 100µM there is a total inhibition of proliferation observed. (Sharma et al., 2007) Figure 2.3.1.1(b) shows the cell cycle analysis based on total cellular DNA content, in this case however CHL concentration of 10µM and 50µM give almost the same daughter cell proliferation. (Sharma et al., 2007) When 100µM CHL were used the reduction in daughter cell proliferation was not totally inhibited as it can be seen from Figure 2.3.1.1(a) (Sharma et al., 2007)

Figure 2.3.1.2 Response to CON A in spleen cells obtained from CHL treated (50, 100 and 200µg/gbw) mice: (a) 4h (b) 24 h and (c) 48h after injection of CHL. Tritiated thymidine ( 1µCi) was added to each well after 72h and cells were harvested 16h later. Each value represents mean counts per minute. (Sharma et al., 2007)

From Figure 2.3.1.2 it can be seen the Con A induced proliferation of lymphocytes obtained at different time interval stated after administration of different doses of CHL. (Sharma et al., 2007) Note the difference in y axis scale of the three graphs from Figure 4.1.2. Figure 4.1.2(a) shows a significant decrease in response to Con A in lymphocytes after 4h CHL was administrated. From Figure 2.3.1.2(b) after 24h of CHL administration the decrease in proliferation of lymphocytes was maximum compared to 4h and 48h of CHL administration. The author also reported that treatment with 200µg/gbw CHL lead to 80% decrement in proliferation of the lymphocytes. (Sharma et al., 2007)

Figure 2.3.1.2(c) shows the increment of cell proliferation after 48h as compared with a lower proliferation for 24h and mice treated with 200µg/gbw in this group did not restored to the control level. (Sharma et al., 2007)

Figure 2.3.1.3 Upregulation of bcl-2 and bcl-xL expression in lymphocytes of CHL treated mice. RT-PCR analysis of (a) bcl-2 and (b) bcl-xL was carried out using lymphocytes isolated from medium and CHL (200µg/gbw, 72h) treated mice. Total RNA from 0.5 x 106 cells was reverse transcribed and PCR amplified with specific primers. PCR products were resolved on 2% agarose gels containing ethidium bromide. β-actin gene expression in each group was used as an internal control. (c) Ratio of intensities of bcl-2 or bcl-xL band to that of respective β -actin band as quantified from gel pictures (Sharma et al., 2007)

The bands appearing at 409, 550 and 556bp on the agarose gel from Figure 2.3.1.3(a) and Figure 2.3.1.3(b) correspond to the mRNA of β –actin, bcl-2 and bcl-xL respectively. It was clear to be seen from the gel that expression of bcl-2 gene was significantly higher in cells treated with 200 µg/gbw CHL for 72h compared to the cells from the medium treated mice. Although the mRNA expression of the medium treated mice lymphocytes do show some faint band. However for bcl-xL only treated cells shows band hence not detectable the expression activity of the gene while untreated mice shows no band in the mRNA expression. Both bcl-2 and bcl-xL are anti-apoptotic gene and the present of the expression of the gene under CHL treatment shows that chlorophyllin prevent the apoptosis of lymphocytes cell and hence prevent immunosupression. This is a very important activity of chlorophyllin as it helps to protect against invaders and hence less susceptible to cancer formation

In this study a lot have been observed such as the lymphocytes proliferation, spleen cell multiplicity and so on. It was observed that chlorophyllin inhibit the in vitro lymphocyte proliferation induced by Con A in a dose dependent manner where CHL exceeded the concentration of 50µM will reduce the proliferation of lymphocytes (Sharma et al., 2007) It was also possible that the reduction in the proliferation of the cells caused by CHL could have been compensated for by prevention of death. It could not be decided whether the reduction in proliferation are due to the activity of apoptosis, further studies and research needed to be done for further explanation. (Sharma et al., 2007) CHL also inhibit the activation of induced cell death program (apoptosis) in Con A stimulated spleen cell as we can see from Figure 2.3.1.3 as the anti-apoptotic gene blc-2 and bcl-xL is up regulated and so apoptosis is prevented (Sharma et al., 2007)

It was reported the activity of CHL to inhibit the proliferation of lymphocytes have the similar action of well- known activity like Vitamin E and gallic acid (Gao et al., 2001). It can be concluded that chlorophyllin do act as a potent anti apoptotic regulator in immune cell system which prevent the apoptosis of lymphocyte when there is inducer such as Con A. However further studies needed to be done to ensure this effect and up regulation of gene is applicable to other animal as well.

2.3.2 Dietary chlorophyllin inhibits the canonical NF-ĸB signaling pathway and induces intrinsic apoptosis

In this section it will be reviewed the activity of chlorophyllin in inducing apoptosis in cancerous/tumor cell. The mechanisms of the pathway involved in induces the apoptosis also the regulation of the gene by chlorophyllin.

In all the cells there is a transcription factors known as the nuclear factor kappa B (NF-ĸB). The function of this transcription factors include controlling multiple cellular processes in cancer, proliferation, inflammation, chemoresistance and radioresistance. (Chatuverdi et al., 2011) In cancerous cell, this transcription will be consecutively active; hence, there will be constitutively activation of genes that control the transcription factors. In unstimulated cell, the transcription factors are switched off , NF-ĸB exist as a heterodimer of p50 and p65 which were sequestered by the inhibitor of NF-ĸB (IĸB) and the activation of NF-ĸB requires the phosphorylation of IĸB-α by IKKβ . This activation of NF-ĸB causes transactivation of 400 genes involved in cell proliferation and apoptosis. (Chatuverdi et al., 2011) There are few proteins that mediate apoptosis evasion in cancer cells are among the most important target of NF-ĸB.

Among the genes that are involved in NF-ĸB pathway, Bcl-2 family protein plays an important role in regulation apoptosis. Bax protein, a pro-apoptotic protein activates the caspase cascade by creating pores in the mitochondrial membrane with the release of cytochrome c another component involved in apoptosis. While Bcl-2, Bcl-xL and Mcl-1 protein inhibits mitochondrial apoptotic pathway hence inhibit and stopped the release of cytochrome C and Smac/DIABLO into the cytosol. Smac, is a mitochondrial proapoptotic protein which promote apoptosis by neutralizing the effect of inhibitor of apoptosis family (IAPs). Another protein, surviving, a family member of the IAPs shuttles between the cytosol and nucleus and is rapidly released when apoptosis occurs (Altieri, 2010).

For the preparation for this study male Syrian hamster 8-10 weeks old with 100 – 110g mass is used as the specimen. The animals were maintained in a controlled environment that is appropriate. Experimental diets were prepared everyday by mixing chlorophyllin to a pre-weighted standard pellet diet (Thiyagarajan et al., 2012). The animals were randomized into six groups with one control group and five experimental groups with each group contain 8 animals. For group 1 animal, the right buccal pouches of hamster were painted with 0.5% of DMBA three times per week for 14 weeks. . For group 2- 4 same treatment were given except each group received additional basal diet containing chlorophyllin at the concentration of 1, 2, 4mg/kg body weight. Group 5 animal received basal diets of chlorophyllin of 4mg/kg body weight without DMBA painted on the buccal pouches. Lastly group 6 animal act as a control.

The experiment terminate after 14 weeks and the buccal pouch were obtained after the hamsters were sacrificed. Following the buccal pouches were then inspected for tumor and the gene expression and protein expressed. Semi-quantitative RT-PCR was used to identify the mRNA in the buccal pouch. SDS-PAGE and Western blot analysis were use to determine the protein expression on the buccal pouch of the hamster.

Table 2.3.2.1 Body weight, tumor incidence, tumor multiplicity, and tumor burden in control and experimental animals. (Thiyagarajan et al., 2012)

Above table shows the result obtained after 14 weeks of experiment conduction. From the table information that can be obtain is that chlorophyllin concentration of 4mg/kg body weight gives the total inhibition of tumor incidence as can be seen from group 4 animal model. While 1mg/kg body weight of CHL administrated to group 2 reduces the tumor incidence by half. Besides, dietary administration of CHL to animals in group 2 to group 4 significantly increases the mean body weight of the animal models.

Figure 2.3.2.1 Representative photomicrographs of well differentiated SCC of the hamster buccal pouch in groups 1-3 (A) , mild hyperplasia in group 4 (B) and normal puccal pouch histology in groups 5 and 6 (C) (hematoxylin and eosin, 20x). (Thiyagarajan et al., 2012)

This figure clearly shows that there is formation of squamous cell carcinoma in buccal pouches of animal in group 1 to 3 which indicate the formation of cancerous cell. While for group 4 there is mild hyperplasia where there is little cell proliferation due to little apoptosis occur. Group 5 and 6 which did not received any DMBA painting on the buccal pouch shows no abnormal cell growth.

Figure 2.3.2.2 Dose-response effect of chlorophyllin on mRNA expression of NF-ĸB, Bcl-2, Bax, and caspase-9 and -3. (A) Representative RT-PCR analyses of NF-ĸB(p50 and p65), Bcl-2, Bax, Caspase-9, Caspase-3 and β-actin are indicated. (B) Densitometric analysis. (Thiyagarajan et al., 2012)

Although administration of chlorophyllin at 1 mg/kg bw (group 2) did not show any significant differences in the expression of these markers as compared to DMBA induced mice, a dose response effect was observed in the expression of these markers in groups 3 and 4, with 4 mg/kg body weight chlorophyllin exerting more significant effects. There is a trend that can be observed from the mRNA expression of the buccal pouch of mice from different group. As the concentration of CHL in basal diet goes higher there is a trend of down regulation of NF-ĸB, Bcl-2, Bax, mRNA expression and a trend of up regulation of caspase-9 and caspase-3 mRNA expression.

Figure 2.3.2.3 Dose- response effect of chlorophyllin on protein expression of NF-ĸB, Bcl-2, Bax, and caspase-9 and -3. (a) Representative immunoblots of NF-ĸB (p50 and p65) nucleus, Bcl-2, Bax, caspase-9, cleaved caspase-3. Protein samples (50µg/lane) resolve on SDS-PAGE was probed with corresponding antibodies. (B) Densitometric analysis of Western blots. The mean protein expression from control lysates for eight determinants was designated as 100% in the graph. Each bar of the other experimental groups represents the mean protein expression of eight determinants. β-actin was used as loading control. (Thiyagarajan et al., 2012)

The same trend follows for protein expression as in mRNA expression discussed earlier from Figure 2.3.2.2.

Figure 2.3.2.4 Effect of chlorophyllin on the mRNA expression of NF-ĸB, IĸB, Bcl-2, Bax, and cytochrome C in control and experimental animals. (A) Representative RT-PCR analysis of NF-ĸB (p50 and p65), I ĸB, Bcl-2, Bax, Cytochrome C and β-actin are indicated. (B) Densitmetric analysis. (Thiyagarajan et al., 2012)

Figure 2.3.2.4 shows the mRNA expression of NF-ĸB family members and markers of intrinsic apoptosis. A significant increase in the mRNA expression of NF-ĸB (p50 and p65) and Bcl-2 with decreased expression of IĸB, Bax, and cytochrome C was seen in the DMBA painted animals (group 1) compared to untreated control. Dietary administration of chlorophyllin down regulated the expression of NF-ĸB and Bcl-2 and enhanced IĸB, Bax, and cytochrome C expression as compared to group 1. In the animals administered chlorophyllin alone, the mRNA expression of the markers analyzed was not significantly different from that in the untreated controls.

Figure 2.3.2.5 Western blot analysis of the expression of NF-ĸB family members in control and experimental animals. (A) Representative immunoblots of NF-ĸB family members. Protein samples (50µg/lane) resolved on SDS-PAGE were probed with corresponding antibodies. (B) Densitometric analysis. The mean protein expression from control lysats for eight determinations was designated as 100%in the graph. Each bar for the other experimental groups represents the mean protein expression of eight determinants. β-actin was used as loading control. (Thiyagarajan et al., 2012)

Figure 2.3.2.5 shows the Western blot analysis of NF- ĸB family members in the buccal pouch of control and experimental animals. Topical application of DMBA induced over expression of nuclear NF- ĸB (both p50 and p65), IKKβ, p-I ĸB and down regulated the expression of IĸB compared to control. Dietary supplementation of chlorophyllin animals with DMBA (group 4) tremendously decreased the expression of nuclear NF-ĸB (p50 and p65), IKKβ, p-IĸB and enhanced the expression of IĸB compared to group1 hamsters. However, dietary supplementation of chlorophyllin alone to group 5 animals did not significantly alter the expression of NF- ĸB family members analyzed compared to control.

Figure 2.3.2.6 Schematic representation of the potential targets of chlorophyllin for the prevention of DMBA induced HBP carcinogenesis (Thiyagarajan et al., 2012).

Administration of chlorophyllin via dietary inhibits the activation of IKKβ. IKKβ involves in the phosphorylation of the inhibitory protein IĸB which subsequently leads to proteosomal degradation. IKKβ inactivation by chlorophyllin results in suppression of the dissociation of IĸB from the NF-ĸB heterodimer (Thiyagarajan et al., 2012). This inactvation leads to the subsequent nuclear translocation of NF-ĸB heterodimer, and leads to the transactivation of genes such as Bcl-2, Bcl-xL, and survivin involved in apoptosis evasion (Thiyagarajan et al., 2012). The down regulation of expression of the antiapoptotic protein Bcl-2 and Bcl-xL consequently lead to an increase in the Bax/Bcl-2 ratio hence promoting mitochondrial outer membrane permeabilization, consequently lead to of apoptogenetic factors such as cytochrome C, and Smac/DIABLO into the cytosol. Released cytochrome C then forms a complex with Apaf-1 and procaspase-9 which leads to the activation of caspase, PARP cleavage, and cell death (Thiyagarajan et al., 2012). In addition, chlorophyllin also enforces nuclear translocation of surviving thereby increasing the susceptibility to intrinsic apoptosis (Thiyagarajan et al., 2012).

The results of the present study shows that dietary chlorophyllin inhibits phosphorylation of IĸB by IKKβ and its subsequent degradation, thereby sequestering NF-ĸB in the cytosol and prevent constitutively activation of the pathway to inhibit apoptosis. In addition, chlorophyllin also blocks nuclear translocation of NF-ĸB that could prevent transactivation of target genes.

In conclusion, the results of the present study provide evidence that chlorophyllin inhibits the development of DMBA-induced HBP carcinomas by abrogating NF-ĸB signaling and inducing intrinsic apoptosis. Chlorophyllin that can simultaneously inhibit NF-ĸB cell survival pathway and induce apoptotic cell death mechanism is an ideal candidate for cancer chemoprevention.