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Palm Fruit Juice Mitigates AZT Mitochondrial Genotoxicity and Dose- Dependent Cytotoxicity

Palm Fruit Juice Mitigates AZT Mitochondrial Genotoxicity and Dose-
Dependent Cytotoxicity
Osborne AE1, Sanchez JA1*, Solomon M2, Stopa A1, Wangh LJ1, Sambanthamurthi R3 and Hayes KC1
1Department of Biology, Brandeis University, Waltham, MA, 02454, USA
2Molecular Pharmacology and Chemistry, Sloan-Kettering Institute, New York, NY 10065, USA
3Malaysian Palm Oil Board, Kajang, Selangor, Malaysia
*Corresponding author: Sanchez JA, Department of Biology, Brandeis University, Waltham, MA, 02454, USA, Tel: 1-781-736-3111; Fax: 1-781-736-3107; E-mail:
sanchez@brandeis.edu
Received date: September 18, 2014; Accepted date: December 16, 2014; Published date: December 20, 2014
Copyright: © 2014 Osborne AE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Chronic use of 3′-Azido-3′-deoxythymidine (AZT) to treat HIV/AIDS causes mitochondrial dysfunction and the
accumulation of mitochondrial mutations. These toxicities have been attributed to increased oxidative damage,
among other mechanisms. Palm fruit juice (PFJ), also known as oil palm phenolics (OPP), is a water soluble by-
product of oil extraction from the fruit of the oil palm (Elaeis guineensis) that is rich in antioxidants and other
phytochemicals. The capacity of PFJ to mitigate AZT mitochondrial genotoxicity (mutagenesis) as well as dose-
dependent cytotoxicity were measured in cultured HepG2 cells. In the presence of PFJ, AZT-induced mutations
were 35% the number of mutations observed in samples treated with AZT alone in the three regions of the
mitochondrial genome examined (HV2, CO2, and ND1). Co-treatment with PFJ increased cell survival in the
presence of increasing doses of AZT by up to 350%. These effects were not due to degradation or inactivation of
AZT by PFJ. The discovery of the mitigating effects of PFJ provides a potential means of ameliorating AZT-induced
mutations and possibly other long-term negative side effects of long-term AZT use.
Keywords: Mitochondria; LATE-PCR; Light-on/Lights-off probes;
AZT toxicity; Palm fruit juice; Oil palm phenolics; Mutational load
Introduction
The nucleotide reverse transcriptase inhibitor (NRTI) 3-
Azido-3-deoxythymidine (AZT, zidovudine) is widely used in the
developing world to treat HIV/AIDS. AZT treatment, however, results
in toxic side effects, including skeletal and cardiac myopathies,
hyperlactatemia, and peripheral neuropathy [1-4]. Chronic AZT
therapy is also associated with an increased incidence of
mitochondria-related diseases such as type 2 diabetes and neurological
disorders [5-7]. These side effects are secondary to the anti-retroviral
activity of the drug and are consistent with AZT damage leading to
mitochondrial dysfunction [8-10]. Because of its toxicity, AZT has
been replaced by other less harmful NRTIs in most developed
countries. Nonetheless, AZT remains an integral component of highly
active anti-retroviral therapy (HAART) in the rest of the world.
Mitochondrial damage and dysfunction resulting from AZT treatment
remain of great concern in the treatment of HIV/AIDS.
The exact mechanism of AZT toxicity is not known. AZT toxicity
could be due to mitochondrial dysfunction resulting from increased
oxidative stress and/or mitochondria DNA mutations. Many clinical
and experimental studies have shown mitochondrial dysfunction and
increased oxidative stress following AZT treatment both in vitro and
in vivo [11-13]. Consistent with these observations, AZT toxicity in
cell culture, mice, and humans has been mitigated by co-treatment
with individual antioxidants (vitamin C, vitamin E, resveratrol, etc.)
[14-17]. There is now direct evidence that AZT causes mitochondrial
DNA mutations [18-20].
This report describes the use of an antioxidant, palm fruit juice
(PFJ), to mitigate AZT-induced mitochondria mutations and dose-
dependent cytotoxicity in HepG2 cells, the experimental system in
which mitochondrial mutagenesis by AZT was recently reported [18].
Palm fruit juice is a water soluble by-product of oil extraction from the
fruit of the oil palm (Elaeis guineensis) that is rich in antioxidant
phenolics [20,21]. Palm fruit juice has high scavenging activity against
hydrogen peroxide, the main contributor to reactive oxygen species
(ROS) produced in mitochondria [20,22,23]. Palm fruit juice also
exhibits various protective biological effects, including anti-
atherogenic, anti-inflammatory, anti-diabetic, and anti-neoplastic
properties [24-31]. For example, dietary supplementation with PFJ
deters the progression of type 2 diabetes, a disease associated with both
mitochondrial dysfunction and chronic AZT treatment [29,31]. The
results of the present study reveal that PFJ mitigated both AZT-
mutagenesis and dose-dependent cytotoxicity.
Materials and Methods
Detection of Mutations after AZT and PFJ Treatment for 30 Days -
Culture Conditions and DNA Preparation
Cell culture conditions and preparation of the mitochondrial DNA
were the same as previously reported [18] except that HepG2 cells
were also cultured in the presence or absence of 25 μg gallic acid
equivalents (GAE)/ml PFJ (a gift from the Malaysian Palm Oil Board).
Twenty-five micrograms GAE per millilitre PFJ was the highest
concentration that permits normal cell growth in conventional Eagle's
Minimum Essential Medium (EMEM, ATCC Manassas, VA) used in
the original experiments where AZT was shown to be mutagenic to
mitochondria [18]. Buffered culture medium employed in the
cytotoxicity study (see Supplemental Data) was not used for these
AIDS & Clinical Research Osborne et al., J AIDS Clin Res 2014, 5:12
http://dx.doi.org/10.4172/2155-6113.1000400
Research Article Open Access
J AIDS Clin Res
ISSN:2155-6113 JAR, an open access journal Volume 5 • Issue 12 • 1000400
experiments to preserve the original culture conditions used to
identify AZT-induced mutations [18]. The thirty-day incubation time
for the genotoxicity study described here and in the original study [18]
was based on reports that mitochondrial dysfunction in HepG2 cells
caused by the physiologically relevant concentrations of AZT used in
our study (7 uM) requires long-term exposure (4 weeks) to develop
[11].
Mitochondrial DNA was prepared as previously reported [32].
Briefly, one microliter of cultured cells (roughly 1000 cells) was lysed
in 14 µl of Quantilyse [33]. Samples were stored at -20°C.
Cytotoxicity Tests
HepG2 cultures were incubated with increasing concentrations of
AZT (0 µM, 10 µM, 30 µM, 50 µM, 70 µM, 100 µM) in the presence or
absence of 75 µg GAE/ml PFJ in buffered media (see Supplemental
Information). Seventy-five micrograms GAE per millilitre PFJ was
used for these studies because it was the highest concentration of PFJ
that did not arrest the growth of HepG2 cells after 25 day culture
under buffered culture conditions (Supplemental Figure 1). Growth
arrest observed at concentrations above 75 µg GAE/ml PFJ is
characteristic of the known cytostatic activity of PFJ against neoplastic
cells such as HepG2 cells [34].
Figure 1: Palm Fruit Juice ameliorated AZT-induced mtDNA
mutations. Thirty days of AZT treatment (7 μM) also resulted in an
increase in mutations in the mitochondrial genome, while co-
treatment with 25 µg GAE/ml PFJ reduced the number of
mutations. Treatment with PFJ alone did not cause an increase in
mutations. White=Non-treated control, Black=AZT treated,
Grey=PFJ treated, and Striped=AZT and PFJ treatment. *represents
those samples that had a p-value of 0.05 or less.
Cells were grown in 6-well plates at 37°C and 5% CO2 in EMEM
supplemented with 25 mM HEPES, 10% fetal bovine serum (BioWest),
50 units/mL Penicillin G, 50 units/mL Streptomycin, and 0.25 µg/mL
Amphotericin B (HyClone Antibiotic/Antimycotic Solution 100X).
Media and additives were replenished every other day. Palm fruit juice
was sterilized through a 0.2 µm filter before addition to the media. Cell
cultures were initially seeded with 1×105 cells/mL. Every 3-4 days cells
were trypsinized and stained for membrane integrity using Trypan
Blue (Sigma Aldrich, St. Louis, MO). The cells were then counted to
measure cell growth and re-plated in fresh media at 1×105 cells/mL.
Experiments were repeated three times.
LATE-PCR Lights-On/Lights-Off Probe Assay for Mutation
Detection
Reagents, primers, probes, and reaction conditions
Lysed DNA samples were first diluted to the digital or near-digital
level (1-5 copies) as described in Osborne et al. [32]. To avoid
contamination, all PCR preparation was conducted in an amplicon-
free hood in a clean room, all targets were kept separate from PCR
reagents at all times, and PCR machines were kept in a separate room
from the laboratory space as far away from the clean room as possible.
PCR amplicons and products were never handled in the same
laboratory space.
Primers, probes and amplification conditions used for the triplex
LATE-PCR assay for mutation analysis of the mitochondrial hyper
variable 2 (HV2) region of the D-Loop, the cytochrome c oxidase
subunit 2 (CO2) gene, the NADH dehydrogenase, subunit 1 (ND1)
gene were as previously described [18]. A single master mix was used
for all experiments to ensure the reproducibility of the fluorescent
signatures.
Mutation analysis
Mutations were scored as described by Osborne et al.. Briefly, sets
of Lights-On/Lights-Off probes spanning the length of each amplicon
were used to generate fluorescent signatures characteristic of each
amplicon sequence. Changes in a fluorescent signature, i.e., shifts to
lower or higher temperature or appearance or disappearance of peaks
and valleys, relative to a reference signature were indicative of
mutations. To obtain reference signatures for normal sequences, bulk
reactions containing over 1000-copies of mtDNA each were amplified
and the resulting fluorescent signatures were averaged together (for a
more detailed discussion of fluorescent signature analysis see Osborne
et al.). Shifted signatures that had more than two temperature points
outside the confidence intervals of the reference signature were
considered a true shift due to mutation rather than noise. These
objective criteria for scoring mutations eliminated subjective bias
during mutation assessment [18].
Measurement of mtDNA copy number
Measurement of mtDNA copy number was carried out as
previously reported in Osborne et al. [18].
Statistical analysis
Statistical analysis of the cytotoxicity and genotoxicity data was
carried out using Pearson’s chi-square test. The Pearson’s chi-square
test establishes whether there is a significant difference between the
expected and observed frequencies in one or more of the treatment
categories. The test was performed using 95% significance (p-value of
less 0.05).
Results
Palm fruit juice mitigated AZT-induced mtDNA mutations
In order to investigate whether increased oxidative stress caused
by AZT leads to mtDNA mutations, AZT-induced mutations were
measured in HepG2 cells treated for 30 days with 7 μM AZT in the
presence and absence of 25 µg GAE/mL PFJ. Seven micromolar AZT is
the peak plasma concentration in AZT-treated individuals previously
found to induce mutations in HepG2 cells [18]. Three regions from
the mitochondrial genome, HV2, CO2, and ND1, were examined for
Citation: Osborne AE, Sanchez JA, Solomon M, Stopa A, Wangh LJ, et al. (2014) Palm Fruit Juice Mitigates AZT Mitochondrial Genotoxicity and
Dose-Dependent Cytotoxicity. J AIDS Clin Res 5: 400. doi:10.4172/2155-6113.1000400
Page 2 of 5
J AIDS Clin Res
ISSN:2155-6113 JAR, an open access journal Volume 5 • Issue 12 • 1000400
mutations using a multiplex LATE-PCR Lights-On/Lights-Off assay at
the digital or near digital levels [18]. A total of 352 wells with 1-5
genomes of mtDNA (an estimated 1,056 amplicons total) were
evaluated.
AZT treatment alone caused up to a 9-fold increase in the number
of random mutations in all three target genes compared to non-treated
samples (p-values of 8×10-6, 0.0003, and 0.0008 for HV2, CO2, and
ND1 respectively; Figure 1). Palm fruit juice treatment alone was not
mutagenic (Figure 1). Co-treatment with 7 μM AZT and 25 µg
GAE/mL PFJ resulted in 35% the number of random mutations caused
by AZT alone (p-values of 0.005, 0.008, and 0.007 for HV2, CO2, and
ND1 respectively; Figure 1). Measurements of the ratio of nuclear to
mitochondrial genomes in PFJ and AZT co-treated samples showed
that PFJ did not reduce the number of mitochondrial mutations by
simply increasing the number of mitochondrial genomes (data not
shown). These results demonstrate that PFJ ameliorated the mutagenic
activity of AZT.
Palm Fruit Juice mitigated dose-dependent AZT cytotoxicity
in HepG2 cells
To investigate whether PFJ also mitigates AZT dose-dependent
cytotoxicity, HepG2 cultures were incubated with various
concentrations of AZT (0 µM, 10 µM, 30 µM, 50 µM, 70 µM, 100 µM)
in the presence or absence of 75µg GAE/mL of PFJ. Increasing
concentrations of AZT from 0 µM to 100 µM resulted in a progressive
increase in cell death after 6 days in culture (Figure 2). Treatment with
100 µM AZT declined cell viability by 90%. Addition of 75 µg
GAE/mL PFJ resulted in increased protection against AZT-induced
cell death from a minimal 12% improvement at 30 µM AZT to about
350% improvement at 100 µM AZT (p-values of 0.04 and 0.01) (Figure
2). These results demonstrate that inclusion of PFJ in the culture
medium mitigated AZT’s dose-dependent cytotoxicity.
Figure 2: Co-treatment with PFJ reduced AZT dose dependent
cytotoxicity after 6 days in HepG2 cells. Cell viability is plotted
against co-treatment of increasing levels of AZT (0 μM, 10 μM, 30
μM, 50 μM, 70 μM, and 100 μM) in the presence of 75 µg GAE/ml
PFJ. White=AZT treated control and Grey=AZT and PFJ
treated.*represents those samples that had a p-value of 0.05 or less.
The error bars show the standard error.
Palm Fruit Juice did not degrade AZT
A possible explanation of the protective effects of PFJ on
mutagenesis and cytotoxicity is that PFJ simply degraded AZT. This
was not the case because day ten cultures showed that AZT modulated
the cytostatic activity of PFJ. In the absence of AZT 75 µg GAE/mL
PFJ did not arrest HepG2 cell growth after 25 days in culture
(Supplemental Figure 1). In contrast, co-treatment with AZT and PFJ
arrested cell growth after ten days in culture, although cells remained
viable (Table 1). These results showed that PFJ did not inactivate AZT
and that the protective effect of PFJ were due to the activity of PFJ
within HepG2 cells.
AZT (µM)
No PFJ
Day 10 Cell Conc.
(cells/ml)
AZT (µM) +
75µg GAE/ml PFJ
Day 10 Cell Conc.
(cells/ml)
0 7.0 x 105 0 6.9 x 105
10 5.6 x 105 10 5.5 x 105
30 4.5 x 105 30 3.0 x 105
50 2.4 x 105 50 1.8 x 105
70 1.3 x 105 * 70 1.1 x 105
100 Dead 100 1.1 x 105
Table 1: High concentrations of AZT enhance the cytostatic effect of
75 µg GAE/ml PFJ.
Day 6 culture cells were trypsinized and plated in fresh culture
media with various concentrations of AZT in the absence or presence
of 75 µg GAE/ml PFJ at an initial concentration of 1×105 cells/ml. At
day 10, cells were trypsinized again, stained with Trypan Blue, and
counted four times per culture to measure the total concentration of
cells. The results show that the same concentration of cells plated on
Day 6 was recovered on Day 10 for 70 µM AZT and 100µM AZT co-
treated with 75 µg GAE/ml PFJ and that cells remained viable. This
phenomenon is characteristic of the cytostatic effect of 75 µg GAE/ml
PFJ on HepG2 cells observed after Day 20 culture in the absence of
AZT (see Supplementary Figure 1) but occurred 15 days earlier. These
data indicate that high concentrations of AZT enhanced the cytostatic
effect of 75 µg GAE/ml PFJ and that growth-arrested cells in the
presence of PFJ continued to be protected from AZT cytotoxicity. In
the presence of lower concentrations of AZT (30 µM and 50 µM), 75
µg GAE/ml PFJ exhibited slower cell growth suggestive of a partial PFJ
cytostatic effect. *=cells displayed an aberrant morphology (Figure 1).
Discussion
The results presented here demonstrate that addition of PFJ
mitigated both AZT-induced mitochondrial mutagenesis and dose-
dependent cytotoxicity. Since AZT toxicity is related to mitochondrial
dysfunction [8-10], the discovery that PFJ mitigated the genotoxic and
cytotoxic effects of AZT suggests that PFJ exerts a protective effect on
mitochondria. These studies expand the range of protective effects
associated with PFJ [24-30].
Protective effects of PFJ against AZT mutagenesis
AZT treatment increases oxidative stress in cells and causes
mutagenesis and dose-dependent cytotoxicity, but the biochemical
basis for these phenomena remains unclear. Since PFJ is rich in
Citation: Osborne AE, Sanchez JA, Solomon M, Stopa A, Wangh LJ, et al. (2014) Palm Fruit Juice Mitigates AZT Mitochondrial Genotoxicity and
Dose-Dependent Cytotoxicity. J AIDS Clin Res 5: 400. doi:10.4172/2155-6113.1000400
Page 3 of 5
J AIDS Clin Res
ISSN:2155-6113 JAR, an open access journal Volume 5 • Issue 12 • 1000400
antioxidants and many antioxidants are known to modulate Reactive
Oxygen Species (ROS) [23] it is logical to postulate that PFJ suppresses
mutations caused by ROS. However, PFJ only reduced the number of
AZT-induced mutations by 65%, suggesting that only a fraction of the
mutations arise because of an increase in ROS and that the AZT also
acts by ROS independent mechanisms. In fact, two hypotheses have
been proposed to explain AZT-induced mutatagenesis. Mutations
might arise from damage of mitochondrial DNA by ROS generated by
AZT [35-38]. Alternatively, mutations may be due either to deleterious
interaction between AZT and the mitochondrial DNA polymerase or
to changes in nucleotide pools, either of which could alter the fidelity
of mtDNA replication [39,40]. It is also possible that the highest
permissible concentration of PFJ in non-buffered media has a limited
antagonistic capacity.
Regardless of which mechanism is operative, AZT-induced DNA
damage likely results in damage to mitochondrial proteins and this, in
turn, probably further increases ROS production and accelerates the
processes of mitochondrial DNA damage and dysfunction. Thus,
independently of mechanism, the functional benefit of PFJ can be
viewed as delaying the accumulation of mitochondrial DNA damage
and the rate at which mitochondrial dysfunction is manifest.
Protective effects of PFJ against AZT cytotoxicity
The protective effects of antioxidants such as PFJ against AZT
cytotoxicity has not been widely studied [15,41]. Palm fruit juice may
attenuate AZT cytotoxicity by counteracting increased oxidative stress.
Given the complex composition of PFJ [42,43] a simple alternative
hypothesis would be that some component(s) of PFJ might inactivate
AZT in the culture media. The fact that AZT enhanced the cytostatic
activity of PFJ argues against PFJ degrading AZT. Independent studies
also show that AZT remains active in the presence of PFJ [23]. The
cytostatic activity of PFJ appears to arise from changes in gene
expression that lead to G1/S phase arrest [44]. Future studies will
address how AZT interacts with cells to enhance the cytostatic effects
of PFJ and how cell cycle arrest prevents AZT-induced cell death.
Significance
In contrast to other antioxidants used to mitigate AZT toxicity
(such as vitamin E and resveratrol [14,17]) which are defined chemical
species, PFJ is a complex mixture of chemicals. The experimental
system described here could be used to identify the component(s) of
PFJ responsible for the range of protective effects observed. The
biomarkers used to evaluate the protective effects of PFJ (cytotoxicity
and mitochondrial genotoxicity) could be used to assess other drugs
and naturally derived phenolics for their effects on mitochondria.
The earlier findings that AZT treatment causes mutagenesis of
mtDNA suggests that patients chronically treated with this drug as
part of HAART might be experiencing continuous damage to their
mitochondrial genomes [18,20,45]. Accumulation of mtDNA damage
over time may explain the increased incidence of mitochondrial-
related diseases observed in these patients. The discovery that PFJ
mitigates AZT toxicity suggests a possible approach to ameliorate or
significantly delay the toxic side effects of AZT affecting millions of
people.
Acknowledgements
This work was funded in part by the Malaysian Palm Oil Board to
K.C.H and L.J.W. The Malaysian Palm Oil Board had no role in study
design; in the collection, analysis, and interpretation of data; in the
writing of the report; and in the decision to submit the paper for
publication.
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Citation: Osborne AE, Sanchez JA, Solomon M, Stopa A, Wangh LJ, et al. (2014) Palm Fruit Juice Mitigates AZT Mitochondrial Genotoxicity and
Dose-Dependent Cytotoxicity. J AIDS Clin Res 5: 400. doi:10.4172/2155-6113.1000400
Page 5 of 5
J AIDS Clin Res
ISSN:2155-6113 JAR, an open access journal Volume 5 • Issue 12 • 1000400
... In agreement with a previous report [7], HepG2 cells treated with a mutagenic concentration of AZT (7 µM) for thirty days developed higher levels of reactive species compared to untreated cells (Table 1). Palm fruit juice treatment alone, which is not mutagenic [23], did not increase reactive species above background. Despite using a concentration of PFJ that mitigated mtDNA mutations [23], AZT-induced reactive species remained elevated in cells cotreated with AZT and PFJ (Table 1). ...
... Palm fruit juice treatment alone, which is not mutagenic [23], did not increase reactive species above background. Despite using a concentration of PFJ that mitigated mtDNA mutations [23], AZT-induced reactive species remained elevated in cells cotreated with AZT and PFJ (Table 1). These results uncouple PFJ mitigation of AZT-induced mtDNA damage from the ability of PFJ to mitigate reactive species generated by AZT. ...
... Depletion of damaged mtDNA could explain the scarcity of oxidative mutations in the AZT mutational spectra. However, previous publications showed that neither AZT treatment, nor PFJ co-treatment, altered mtDNA copy number [4,23]. Thus, selective loss of mutated mtDNA would not account for the relative lack of signature mutations for oxidative damage in these experiments. ...
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Addition of clinically-relevant levels of 3′-Azido-3′-deoxythymidine (AZT) to cultured HepG2 cells increases the number of reactive radical species (reactive oxygen and nitrogen species [ROS and RNS]) as well as random mutations in mitochondrial DNA (mtDNA). Co-treatment of AZT-exposed cells with palm fruit juice (PFJ) mitigates AZT mutagenesis. These findings suggest that AZT-dependent mtDNA damage resulted from increased reactive species and that PFJ, a known anti-oxidant, mitigated such damage by decreasing the levels of these species. The present report tests the predictions that (1) PFJ mitigates AZT mutagenesis by reducing the burden of AZT-induced reactive species, and (2) AZT-induced mutations in mtDNA should predominantly consist of G → T and C → A substitutions characteristic of DNA oxidative damage. Levels of reactive species and mitochondrial mutagenesis were measured in HepG2 cells exposed AZT in the presence or absence of PFJ. Controls experiments showed that PFJ in HepG2 cells exhibited strong scavenging activity against hydrogen peroxide-induced ROS, the main reactive species generated by dysfunctional mitochondria. Despite this strong antioxidant activity PFJ did not decrease AZT-induced reactive species at a concentration that mitigated mtDNA mutations. Consistent with this observation, the spectrum of AZT-induced mutations did not fit the spectrum expected from direct mtDNA oxidative damage. Instead, the spectrum obtained was consistent with the majority of mutations (80%) arising from mitochondrial DNA polymerase errors induced by AZT. These observations suggest that oxidative damage was not the major contributor to AZT-induced mutations.
... The left 3 columns were taken from rats without PFB supplementation and show faint basal staining for TH. The right 2 columns are from NGR supplemented with PFB and show strong dark 50 Reduction in arterial blood pressure via increased endothelial production of nitric oxide Rabbit Sambanthamurthi et al. 65 Reduction of reactive oxygen species via direct neutralization and increased oxido-reductases In vitro Sambanthamurthi et al. 65 Inhibition of cancer cell proliferation and growth; inhibition of proliferation and growth of xenotransplanted tumors Mouse Sambanthamurthi et al. 51 Sekaran 77 Ji et al. 78 Reduction in retroviral replication via inhibitory action against HIV protease and reverse transcriptase In vitro Sambanthamurthi et al. 79 Modulation of IL-1β-activated astrocyte cytokine secretome with reduction of TNF-α, RANTES, IP-10 In vitro Weinberg et al. 54 Reduction in beta-amyloid (1-42) peptide aggregation and reduced fibrillization In vitro Weinberg et al. 53 Reduction in mitochondrial genome mutation rate following exposure to genotoxins In vitro Osborne et al. 80 Inhibition of angiogenesis and inflammation Mouse Zandi et al. 52 Content courtesy of Springer Nature, terms of use apply. Rights reserved www.nature.com/scientificreports ...
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... OPP has also been shown to lower the immune response of tumor bearing mice compared to controls, suggesting delayed inflammation in OPP supplemented mice [86]. PFJ has been shown to offset oxidative stress and 3 -Azido-3 -deoxythymidine (AZT)-induced mitochondrial genotoxicity (mutagenesis) and dose-dependent cytotoxicity in HepG2 (a human liver cancer cell) cells suggesting that PFJ exerts a protective effect on the mitochondria [87]. This may be of particular importance for age related changes associated with inflammaging which have been associated with increases in mitochondrial prooxidants. ...
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Cognitive function is a key aspect of healthy aging. Inflammation associated with normal aging, also called inflammaging is a primary risk factor for cognitive decline. A diet high in fruits and vegetable and lower in calories, particularly a Mediterranean Diet, may lower the risk of age-related cognitive decline due in part to the associated high intake of antioxidants and polyphenols. A phenolic, Palm Fruit Bioactive complex (PFBc) derived from the extraction process of palm oil from oil palm fruit (Elaeis guineensis), is reported to offset inflammation due to its high antioxidant, especially vitamin E, and polyphenol content. The benefit is thought to be achieved via the influence of antioxidants on gene expression. It is the purpose of this comprehensive review to discuss the etiology, including gene expression, of mild cognitive impairment (MCI) specific to dietary intake of antioxidants and polyphenols and to focus on the potential impact of nutritional interventions specifically PFBc has on MCI. Several in vitro, in vivo and animal studies support multiple benefits of PFBc especially for improving cognitive function via anti-inflammatory and antioxidant mechanisms. While more human studies are needed, those completed thus far support the benefit of consuming PFBc to enhance cognitive function via its anti-inflammatory antioxidant functions.
... The phenolic compounds include phydroxybenzoic acid and three caffeoylshikimic acid isomers (Sambanthamurthi et al., 2011a). PFJ has demonstrated positive effects against degenerative diseases in various biological models without toxicity (Abeywardena et al., 2013;Bolsinger et al., 2014;Che Idris et al., 2014;Ji et al., 2015;Osborne et al., 2014;Patten et al., 2015;Sambanthamurthi et al., 2011a;Sambanthamurthi et al., 2011b;Sekaran et al., 2010). The No Observable Adverse Effect Level (NOAEL) of PFJ was considered to be 2000 mg/kg body weight/day (Lynch et al., 2017). ...
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Addition of clinically-relevant levels of 3′-Azido-3′-deoxythymidine (AZT) to cultured HepG2 cells increases the number of reactive radical species (reactive oxygen and nitrogen species [ROS and RNS]) as well as random mutations in mitochondrial DNA (mtDNA). Co-treatment of AZT-exposed cells with palm fruit juice (PFJ) mitigates AZT mutagenesis. These findings suggest that AZT-dependent mtDNA damage resulted from increased reactive species and that PFJ, a known anti-oxidant, mitigated such damage by decreasing the levels of these species. The present report tests the predictions that (1) PFJ mitigates AZT mutagenesis by reducing the burden of AZT-induced reactive species, and (2) AZT-induced mutations in mtDNA should predominantly consist of G → T and C → A substitutions characteristic of DNA oxidative damage. Levels of reactive species and mitochondrial mutagenesis were measured in HepG2 cells exposed AZT in the presence or absence of PFJ. Controls experiments showed that PFJ in HepG2 cells exhibited strong scavenging activity against hydrogen peroxide-induced ROS, the main reactive species generated by dysfunctional mitochondria. Despite this strong antioxidant activity PFJ did not decrease AZT-induced reactive species at a concentration that mitigated mtDNA mutations. Consistent with this observation, the spectrum of AZT-induced mutations did not fit the spectrum expected from direct mtDNA oxidative damage. Instead, the spectrum obtained was consistent with the majority of mutations (80%) arising from mitochondrial DNA polymerase errors induced by AZT. These observations suggest that oxidative damage was not the major contributor to AZT-induced mutations.
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