|Year : 2019 | Volume
| Issue : 3 | Page : 216-221
Antioxidant and chemotherapeutic effects of trèvo®supplement on benzene-induced leukaemia in murine models
Oluwanishola Z Shehu1, Olufemi E Akanni1, Muhammed R Shehu2, Kamoru A Adedokun3, Ramat T Kamorudeen4
1 Department of Medical Laboratory Science, College of Health Sciences, Ladoke Akintola University of Technology, Osogbo, Osun State, Nigeria
2 Department of Microbiology, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia
3 Department of Oral Pathology, DUH, King Saud University Medical City, Riyadh, Kingdom of Saudi Arabia
4 Children Welfare Unit, Osun State Hospital Management Board, Asubiaro, Osogbo, Osun State, Nigeria
|Date of Web Publication||6-Sep-2019|
Olufemi E Akanni
Department of Medical Laboratory Science, College of Health Sciences, Ladoke Akintola University, Ogbomoso, Oyo State
Source of Support: None, Conflict of Interest: None
Introduction: Oxidative stress is largely implicated in a molecular mechanism involving initiation, development and progression of leukaemogenesis. Trévo® supplement is a multiherbal formula produced from various phytonutrients with antioxidant potential. We investigated the antioxidant activities of Trèvo® supplement as a prospect for leukaemia treatment. Materials and Methods: The study was carried out on 36 Wistar rats weighing between 140 g and 160g. They were randomly divided into six groups. Group 1 (positive controls) were induced with 0.2 ml benzene chromosolv solution 48 hourly for 4 consecutive weeks and fed with rat pellets without Trévo® supplement. Group 2 (negative controls) received only rat pellets. Group 3 received only normal dose of Trévo® supplement with rat pellets. Groups 4, 5 and 6 were induced for 4 weeks followed with low-, moderate- and high-dose Trévo® supplement for 3 weeks with rat pellets, respectively. Glutathione (GSH), catalase (CAT), malondialdehyde (MDA), total protein (TP) and gamma-glutamyltransferase (GGT) plasma concentrations were assayed simultaneously. Results: Induction of leukaemia was evidenced in positive controls by elevated total white blood cell counts (marked with lymphocytosis) and mild anaemia with reduced haemoglobin counts. Furthermore, GSH, CAT and TP levels for graded dosages of Trévo®-treated groups (following benzene induction) showed statistically significant elevations (P < 0.05) compared to the benzene-induced positive controls, whereas MDA and GGT levels with high-dose Trévo® treatment showed statistically significant reductions (P < 0.05) compared to the positive controls. Conclusions: Trévo® supplement exhibited profound antioxidant potential indicated by improvement from leukaemia after oral administration. This amelioration is believed to be associated with the nutritional supplement in a dose-dependent manner.
Keywords: Antioxidant, chemotherapy, leukaemia, oxidative stress, phytonutrients, Trévo® supplement
|How to cite this article:|
Shehu OZ, Akanni OE, Shehu MR, Adedokun KA, Kamorudeen RT. Antioxidant and chemotherapeutic effects of trèvo®supplement on benzene-induced leukaemia in murine models. Adv Hum Biol 2019;9:216-21
|How to cite this URL:|
Shehu OZ, Akanni OE, Shehu MR, Adedokun KA, Kamorudeen RT. Antioxidant and chemotherapeutic effects of trèvo®supplement on benzene-induced leukaemia in murine models. Adv Hum Biol [serial online] 2019 [cited 2020 Jan 25];9:216-21. Available from: http://www.aihbonline.com/text.asp?2019/9/3/216/266220
| Introduction|| |
Reactive oxygen species (ROS) are highly chemically reactive molecules which play a vital role in building up oxidative stress (OS) in the body systems. OS develops when there is an abnormality in oxidative–antioxidative balance in the body. Effects of abnormalities in the oxidative–antioxidative imbalance in the biological systems have been repeatedly reported in different haematologic malignancies, including acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia and myelodysplastic syndrome.,, In addition, report shows that leukaemic cells generate an elevated level of ROS more than non-leukaemic cells. A separate report shows that OS is implicated in leukaemogenesis through regulations of cell proliferation and cell signalling. Again, the study shows that OS may prompt the development of leukaemia mechanistically by inducing many oncogenic pathways, involving Ras and vascular endothelial growth factor and the nuclear factor-κB through signal transduction.
Meanwhile, the main available treatment for the management of leukaemia is high-dose cytotoxic chemotherapy, which may also trigger more detrimental ROS and thus sets off OS. Induction of OS by chemotherapy has been reported in leukaemia treatments. Providentially, due to the associated side effects of chemotherapy, compounds from nutraceuticals and natural derivatives were recently anticipated. Emerging studies suggest that natural compounds contain novel and biologically active compounds which are able to impact cellular and molecular processes underlying tumour progression and limit the side effects commonly associated with cancer treatment.,,
Interestingly, Trèvo formula contains potential antioxidants according to the manufacturer. Trèvo dietary formula is a multiherbal concoction manufactured under the trade name TRÉVO (TRÉVO LLC™) by United Int'l Lab LLC, TX 75244, USA. The constituents involve many ingredients from phytonutrients of common garden fruits and vegetables, herbs and coral calcium complex containing Vitamins (A, C and E), amino acids, trace minerals, essential fatty acids, digestive enzymes and coenzymes, apart from primary essential antioxidants. It is an established fact that micronutrients are essential components of antioxidant system in the biological systems. These products may, therefore, obviate possible OS from free radicals generated consequent of leukaemogenesis or during treatment of the disease, owing to their possible antioxidant properties in the constituents of Trévo formula. In addition, the drug intolerance associated with leukaemia chemotherapy due to elevated ROS production calls for new therapeutic approach. This study thus assessed the antioxidant potential of this formula in the treatment of leukaemia.
| Materials and Methods|| |
This is an interventional experimental study consisting of 36 Wistar rats weighing between 140 g and 160 g that were randomly divided into six groups containing six animals per group (in two replicates of three animals each).
Experimental protocol and animal treatments
- Group 1 – Positive control, PC (benzene induction for 4 weeks, no supplement treatment and rat pellets)
- Group 2 – Negative control, NC (no benzene induction, no supplement and rat pellets only)
- Group 3 – Non-induced supplemented diet group, TGo(no benzene induction, normal dose supplement for 4 weeks and rat pellets)
- Group 4 – Test Group 1, TG1 (benzene induction for 4 weeks, low-dose supplement for 3 weeks and rat pellets)
- Group 5 – Test Group 2, TG2 (benzene induction for 4 weeks, moderate-dose supplement for 3 weeks and rat pellets)
- Group 6 – Test Group 3, TG3 (benzene induction for 4 weeks, high-dose supplement for 3 weeks and rat pellets).
Administration of benzene solution
Benzene chromosolv solution is a product of Sigma-Aldrich (with Cat No 270709; ≥99.9% conc.). 0.2 ml was administered through the vein of the tail 48 hourly for 4 weeks under controlled monitoring.
Major ingredients and chemical composition of Trévo dietary formula
The major ingredients consist of 13 essential vitamins, 20 various amino acids, 14 trace minerals, coenzyme Q10, 18 different vegetables, fatty acids of plant source, 5 green super foods and coral calcium (1000 mg)., The phytochemical analysis shows the presence of polyphenols, flavonoids, alkaloids, phlobatannin, tannin and saponin.
Administration of Trèvo supplement
Calculations for Trèvo supplement dosage
For an average adult (70 kg), the dose is 2 ounces ≈ 52 ml, i.e., 1 ounce = 26 ml.
70kg → 52 ml
1kg → X
X = 52/70 = 0.7 ml/kg.
Average weight of rats is 150 g ≈ 0.15 kg
Since 1 kg → 0.7ml
0.15 kg → X
X = 0.7 × 0.15 = 0.105 ≈ 0.11 ml
Therefore, the normal dose for normal rats (i.e., non-induced, with supplemented diet group, TGo) is 0.11 ml.
For the treatment of leukaemia, the recommended dose is 5 ounces ≈ 130 ml.
70 kg → 130 ml
1 kg → X
X = 130/70 = 1.86 ml.
Average weight of rats is 0.15 kg,
1 kg → 1.86 ml
0.15 kg → X
X = 0.15 × 1.86 = 0.3 ml.
Therefore, moderate dose for test Group 1, TG1 =0.3 ml/kg.
Low dose for test Group 2, TG2 =0.3/2 = 0.15 ml/kg.
High dose for test Group 3, TG3 =0.3 × 2 = 0.6 ml/kg.
Method of Trèvo supplementation
Subsequently after the injection of benzene, Trèvo supplement (with approval no A7-1020 of the National Agency for Food and Drug Administration and Control) was administered orally once daily at low (0.15 ml/kg), moderate (0.3 ml/kg) and high dose (0.6 ml/kg) for 3 weeks. This followed the experimental protocol using 0.11 ml, 0.3 ml, 0.15 ml and 0.6 ml for groups TGo, TG1, TG2 and TG3, respectively. Trèvo formula was obtained from a registered distributor in Osun State, Nigeria.
Blood sample collection
Blood samples were collected into EDTA and plain bottles by heart puncturing after 12 h of the last treatment. The liver was removed, weighed and fixed in 10% formalin. 1 g of the liver was washed with 5 ml of buffer and homogenised with mortar and pestle in 4 ml of homogenising buffer and finally stored at −20°C until the time of analysis.
The auto analyser (Sysmex) was used to measure haematocrit (HCT), total WBC count, red blood cells count, haemoglobin (HGb), platelets counts, absolute lymphocytes count, absolute mixed count and absolute neutrophil count using blood collected in EDTA anticoagulant (50 μl).
Other biochemical analyses
Estimation of malondialdehyde level
To estimate malondialdehyde (MDA) activity, a modified thiobarbituric acid reactive substances assay was adopted. The measurement of the product of the lipid peroxidation (MDA) followed Ahmad et al.'s method using liver homogenate as lipid-rich medium. 0.4 ml of the homogenised sample was added to 1.6 ml of buffer, 0.5 ml of TCA and 0.5 ml of TBA. The resulting mixture was vortexed and heated at 95°C for 45 min. After cooling, the mixture was read at a wavelength of 532 nm.
Estimation of glutathione level
For estimation of GSH, the method described by Beutler et al. was followed. 0.2 ml of homogenate sample was diluted in 1.8 ml of distilled water. 3 ml of the precipitating reagent (SSA) was added to the diluted sample and then allowed to stand for 10 min. The mixture was centrifuged for 5 min at 3000 g while 0.5 ml of the supernatant was added to a separate 4 ml of phosphate buffer pH 7.4. Finally, 0.5 ml of Ellman's reagent was added to the mixture. The absorbance was read at 412 nm within 30 min of the colour development. GSH standard curve was constructed using serially diluted GSH stock (40 μg/ml) in phosphate buffer. A graph of concentration against the absorbance of the dilutions was plotted.
Estimation of catalase activity
Catalase (CAT) activity was determined according to the method of Sinha. 1 ml of the serum was diluted with 49 ml of distilled water and mixed with the assay mixture (1 ml of H2O2 solution [200 μmole] and 5 ml of phosphate buffer) by a gentle swirling motion at room temperature. 1 ml portion of the reaction mixture was blown into 2 ml dichromate/acetic acid reagent at 60 s intervals and the absorbance taken at 570 nm. The mononuclear velocity constant k for the decomposition of H2O2 by CAT was determined using the equation for a first-order reaction, according to the equation:
K = 1/t log S0/S.
Where, S0 is the initial concentration of the hydrogen peroxide and S is the concentration of the peroxide at time t minute. The values of the k are plotted against time in minutes and the velocity constant of CAT k0 at 0 min determined by extrapolation. The CAT contents of the serum were expressed in terms of cat feiahigkeit or 'cat f'.
Cat f = K0/mg protein (ml).
K0= H2O2 consumed.
H2O2 consumed = 200 – H2O2 remained.
Estimation of total protein concentration
Quantitative determination of total protein (TP) was carried out using biuret (colorimetric) method of Siltanen and Kekki, measured at 540 nm in a matched cuvette and 1.0 cm light path. The CHEMLEX, S. A. laboratory kit (LABKIT) was used. The absorbance (A) of the sample and calibrator were read spectrophotometrically at 540 nm against a blank. The colour was stable for at least 30 min.
Estimation of gamma-glutamyltransferase activity
This was determined using 'LABKIT' as follows: 1000 μl of working reagent was pipette into a clean matched cuvette (with 1.0 cm light path). 100 μl of the sample was added into the cuvette, mixed and incubated for 1 min at room temperature. The absorbance (A) was read at 1 min interval for 3 mins using a spectrophotometer at a wavelength of 405 nm. The difference of absorbance and the average absorbance difference per minute (△A/min) were calculated. U/L of γ-GT = △A/min × 1190.
The Ward linkage using SPSS Statistical Package for the Social Sciences Statistics (IBM® SPSS Statistics; Ver. 22, USA) was used for statistical analysis. Values obtained from the study were expressed as mean ± standard deviation (SD) and compared using analysis of variance, and the significance was measured at P < 0.05.
| Results|| |
[Table 1] shows the result of haematological parameters (means ± SD) in positive (PC) and negative (NC) controls. The white blood cell (WBC) count increased significantly (P < 0.05) with marked lymphocytosis. The haematocrit, haemoglobin concentration (HGB) and red blood cell count (RBC) reduced significantly (P < 0.05). The results indicated a successful leukaemia induction in the PC.
|Table 1: Effects of benzene injection on haematological parameters in positive and negative controls|
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[Table 2] shows the mean (±SD) results of biochemical markers of OS in PC (leukaemia-induced rat) against the healthy rats, non-leukaemia induced and without Trévo supplement (negative control group, NC). Comparisons showed statistically significant differences (P < 0.05) in all the parameters (GSH, CAT, MDA, TP and gamma-glutamyltransferase [GGT]).
|Table 2: Comparison of parameters of oxidative stress in leukaemic rats (positive controls) against the healthy rats (negative controls)|
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[Table 3] shows the mean (±SD) results of biochemical markers of OS in all the experimental groups. Mean values of reduced glutathione (GSH), CAT and TP were low, whereas MDA and GGT were higher in PC group against the other groups (negative group, NC and test groups 1, 2 and 3) which also indicated leukaemia induction.
[Table 4] shows the mean (±SD) of OS parameters in leukaemia-induced rats without supplement (positive group, PC) compared to non-induced group fed with supplemented diet (TG0) and leukaemia-induced treated groups with low dose (TG1), moderate dose (TG2) and high dose (TG3) of Trévo supplement. It was observed that in all the parameters (GSH, CAT, MDA, TP and GGT), TG0 and TG3 showed statistically significant differences (P < 0.05) compared to the PC group. Furthermore, GSH showed statistically significant reduction (P < 0.05) in PC against TG1, whereas both GSH and TP showed statistically significant reductions (P < 0.05) in PC compared to TG2. Although there were reductions in GSH, CAT and TP levels along with high concentrations of MDA and GGT in PC group against all other groups, no statistical differences (P > 0.05) were observed.
[Table 4] shows the mean (±SD) results of OS parameters in healthy rats non–leukaemia induced and without Trévo supplement (negative control group, NC) compared with TG0, TG1, TG2 and TG3. It was observed that in all the parameters (GSH, CAT, MDA, TP and GGT) as opposed to the PC [Table 4], TG0 and TG3 showed no statistically significant differences (P > 0.05) compared to the NC group. Similarly, MDA (in TG1 gr oup), GSH, MDA, TP and GGT (in TG2) all showed no statistically significant differences (P > 0.05) compared with NC. However, there were statistically significant reductions (P < 0.05) in GSH, CAT and GGT levels in TG1 group and also CAT level in GT2 group, respectively.
| Discussion|| |
ROS and lipid peroxide products have been implicated with deleterious effect on genomic and mitochondrial DNA with reference to DNA damage in consequence of oxidative reactivity, resulting in tumourigenesis through replication on mutations., Similarly, the previous report showed that ROS increases the rate of mutation in a murine model causing various forms of leukaemia. Of recent, some natural products and nutraceuticals with potential antioxidant benefits have been reported and envisaged to serve a future purpose in the treatment of some OS-induced disorders.
In this study, lipid peroxidation products and endogenous enzymes with antioxidant properties were used to assess OS in Wistar rats. Initial result, following benzene induction, confirmed a successful induction of leukaemia with the significant proliferation of WBC counts (with marked lymphocytosis), anaemia and thrombocytopaenia when compared to the non-induced group [Table 1]. In line with our study, benzene-induced leukaemia had been reported earlier in both humans and animals with various haematological abnormalities.
Furthermore, in the present study, advance results showed that there was an evidence of OS with reduced GSH level in leukaemic rats [Table 2]. However, further treatment with graded doses of Trévo supplement in other groups of rats was found to boost GSH significantly to a level remarkably similar to what was observed in the healthy rats [Table 3] and [Table 4]. This implied that graded doses of Trévo supplement may have the potential to enhance the level of GSH antioxidant system. The previous study showed that GSH plays a vital role in detoxifying cancer-inducing agents and concluded that an elevated GSH level may alter the oncogenic pathway thus reflecting on cell survival. In agreement with this study, Trèvo supplement had been reported for a protective effect against OS through the elevation of the antioxidative defence enzyme while extensively reducing the level of lipid peroxidation. This effect was conclusively attributed to the elevation of GSH level and other potent antioxidants Vitamins - C, E, A and bioflavonoids.
Again, CAT, an enzymatic antioxidant widely distributed in the body, is known for the protective role against reactive hydroxyl radicals. Reduction in CAT activity had been reported in a number of deleterious effects due to ineffective assimilation of superoxide radical and hydrogen peroxide. Previously, reduced CAT level was reported as a common occurrence in leukaemias. In agreement with our result, the present study showed a significant reduction in CAT level in leukaemic rats compared to the healthy ones [Table 2] and [Table 3]. Interestingly, other groups of leukaemic rats further treated with graded doses of Trévo supplement showed increases in CAT levels compared to the leukaemic rats without any treatment. However, it is noteworthy to state categorically that only high-dose treatment was able to attain a considerable boost in CAT levels in leukaemia-induced rats to a remarkable level similar to what was observed in the normal/healthy group [Table 3] and [Table 4]. Hitherto, Trévo supplementation at high dose showed great potential in restoring CAT activity in the leukaemic rats.
In addition, MDA is a product of lipid peroxidation and popularly signifies a specific biomarker of OS and disease progression, often times implicates mutagenic and carcinogenic activities. The roles of MDA have been overemphasised repeatedly in a number of cancers, including solid tumours and leukaemias., We assessed the MDA level as a clinical biomarker of OS in leukaemia-induced rats. MDA level was found appreciably high compared to the healthy rats. This indicated that leukaemic rats showed OS response through lipid peroxidation. However, in the treatment groups, following induction, MDA levels were reduced in a dose-dependent manner with graded doses of Trévo supplement, even though significant difference was only recorded in high-dose treatment compared to the leukaemic group (without any treatment) [Table 4]. Thus, this suggests that Trévo supplementation at various doses may possess effective recovery capacity after the development of leukaemia in the rats. In other words, our findings showed that Trévo supplement could be a potent antioxidant formula capable of preventing and reversing oxidative damage by attenuation of OS. This result corroborates several reports using dietary supplementation or formula in the treatment of cancers and other disorders, specifically by reducing MDA levels.,
Moreover, the present study showed that the TP level in leukaemic rats was significantly reduced compared to the healthy rats. Similarly, significant increases in TP were observed in moderate and high doses of Trévo-supplemented treatment, whereas low dose showed no considerable difference when compared to the leukaemic rats (without treatment). As a result, Trévo supplement showed nutritional value through enhanced protein synthesis with restorative influence dose dependently, all attributable to the supplementation in the treated groups. TP constitutively consists of albumin and globulins in the blood system. A previous study linked patients suffering from leukaemia under treatment with regaining albumin and alpha globulin values toward normal blood levels. On this background, we suggest that increased TP in the present study could be due to recuperation from leukaemia and hepatotoxic injury after supplementation. In concordance with the present study, earlier report associated benzene exposure with haematologic and hepatic abnormalities.
Furthermore, γ-GT/GGT is an enzyme responsible for the extracellular catabolism of GSH, the main thiol antioxidant in mammalian cells. GGT is commonly used as a diagnostic biomarker to assess liver dysfunction. In this study, GGT level was found considerably increased in the leukaemic group when matched against the healthy group [Table 3] and [Table 4]. This elevated GGT level could be secondary to hepatic injury in the leukaemic group in consequence of exposure to OS. However, GGT level of leukaemic groups treated with graded doses of Trévo supplement had reduced levels of GGT in a dose-dependent manner, whereas only high-dose treatment showed a marked difference compared to the leukaemic group (without supplement) as shown in [Table 3] and [Table 4]. Our result is, therefore, in agreement with a study in Makkah, Saudi Arabia, which demonstrated oxidant hepatic and haemotoxic injuries on gasoline workers exposed to benzene vapour, followed with recovery on the administration of supplemented micronutrients as antioxidant agents.
| Conclusions|| |
The present study demonstrated high-antioxidant activity of Trèvo supplement with both chemopreventive and chemotherapeutic potentials. The antioxidant treatment is capable of terminating the free radical process and may be considered as a possible protection agent in subverting the progress of oxidative damage in the biological systems, particularly involving leukaemia and also possibly from haematotoxic and hepatotoxic injuries. In addition, Trévo supplement may be formulated as a potential targeted OS therapy, especially when combined with the existing cytotoxic chemotherapy to offer improved synergistic effects for various haematological malignancies in the future.
We would like to thank the authority of Ladoke Akintola University of Technology (LAUTECH), Ogbomoso, Oyo State, Nigeria, for providing some of the facilities used in these studies. We also appreciate the technical assistance provided by Messrs. Olaniran O. I. and Adegoke O. of pharmacology and therapeutic department and animal house LAUTECH, respectively, as well as the support by our research assistants, is also appreciated.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]