Medical Hypotheses 88 (2016) 9199

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Medical Hypotheses
journal homepage: www.elsevier.com/locate/mehy

Human gastric cancer, Helicobacter pylori and bracken carcinogens:

A connecting hypothesis
Alberto Oliveros-Bastidas, Mara Pa Calcagno-Pissarelli, Marlene Naya, Jorge Luis vila-Nez,
Miguel E. Alonso-Amelot
Chemical Ecology Group, Faculty of Sciences, University of Los Andes, Mrida 5101, Venezuela

a r t i c l e i n f o a b s t r a c t

Article history: Long term infection of Helicobacter pylori (Hp) virulent strains is a key factor in the genesis of human gas-
Received 21 June 2015 tric cancer, and so are certain dietary proinflammatory and genotoxic compounds. Carcinogenic bracken
Accepted 8 November 2015 fern (Pteridium spp.) is one of these. Toxins from this plant are consumed as bracken culinary prepara-
tions, through milk and meat of bracken-exposed livestock, and drain waters from bracken swards.
Bracken toxin ptaquiloside (PtQ), a suspected human carcinogen, elicits complex responses in animals
leading to death. PtQ and Hp might cooperate in gastric pathologies. This paper presents an hypothesis
on PtQHp association leading to the enhancement of carcinogenesis in the human gastric environment
that might explain the high gastric cancer incidence and death rates among Hp-infected people living in
bracken zones at two levels: (1) The macroscopic scale comprising the flow of PtQ in the human diet. (2)
the microscopic scale encompassing (A) gastric luminal medium; (B) gastric mucus structure and mucin
degradation elicited by Hp; (C) bacterial pH gradient modification of the gastric mucosa that favors PtQ
survival and its penetration into epithelial tissue; (D) combined PtQ/Hp effects on gastric immune and
inflammatory responses; (E) PtQHp complementary activity at selected cell signaling cascades and gen-
ome disturbance.
2015 Elsevier Ltd. All rights reserved.

Introduction Bioactive dietary organic compounds including carcinogens

Human gastric cancer (HGC) continues to be a primary cause of
death among all malignant neoplasms, despite some improve-
ments in recent years [1]. Incidence rates are unevenly distributed,
being substantially higher in Asian and most developing countries
where HGC amounts to two thirds of all new cases [2].
HGC is a multifactorial inflammatory disease of great molecular
complexity [3] in which overexpression of several inflammatory
proteins, numerous gene transcription alterations and mutations
occur [4]. A suit of contributing factors has been identified. Among
non-smokers, the two most conspicuous environmentally causal
agents are Helicobacter pylori (Hp) infection and diet, be it excess
of cancer-prompting foods, lack of cancer-protective victuals or
both [5]. Host genetic background [6], deprivation, illiteracy and
occupation [7] are involved as well. The gastric human environ-
ment exposed to todays profuse dietary offer is exceedingly com-
plex and so ought to be the number and combinations of candidate
components influencing the onset and progress of HGC.
Corresponding author. Tel.: +34 96 578 7578.
E-mail address: alonsome123@gmail.com (M.E. Alonso-Amelot).
gain access to gastric epithelia, where primary adenocarcinoma
generally forms, through two major routes: (1) topic contact with
the apical side of epithelial cells followed by transmembrane trans-
port, and (2) previous gastrointestinal uptake, plasma or lymph
carriage, metabolic derivatization in some cases and transport to
the gastric epithelial cell domain from the vascular side. Hp oper-
ates through route (1) [8], whereas dietary materials utilize both
routes [9]. This predicament creates considerable difficulty to
define carcinogenic mechanisms and thresholds [10]. As a com-
pounding effect, indirect proinflammatory reactions via cytokine
over-expression may be elicited on infiltrating immune cells by
bacteria [11], physical insult and dietary xenobiotics equally [12],
leading to chronic inflammation, excessive production of reactive
oxygen and nitrogen species (RONS) and thereby profound molec-
ular disturbance and cancer.
Against these threats the stomach of vertebrates has erected
effective physical and chemical barriers, including a strongly acidic
lumen, a thick mucus layer with high turnover rates to flush away
xenobiotic material, a bioactive multifunctional epithelial struc-
ture that is actively being replaced from a stem cell reservoir and

http://dx.doi.org/10.1016/j.mehy.2015.11.007
0306-9877/ 2015 Elsevier Ltd. All rights reserved.
92 A. Oliveros-Bastidas et al. / Medical Hypotheses 88 (2016) 9199
a suite of enzymes and nitric oxide to neutralize xenobiotic action
[13].
HGC onset and progression involves a multitude of molecular
transformations spanning several years. This scenario requires a
long term influence of cancer etiological agents. Hp infection,
deep-rooted dietary habits and exposure to persistent environ-
mental pollutants qualify as such agents. Although the best well
studied of these is Hp, attributing HGC to this bacterial species
alone might not be fully granted [1416]. That Hp is a necessary
but not sufficient factor for HGC is gaining increasing consensus.
As a result, carcinogenic insult by Hp might be brought to neoplas-
tic completion by specific compounds in the diet [1719].
Here we raise the following hypothesis: ptaquiloside (PtQ), a
major carcinogen found in ubiquitous bracken fern (Pteridium
spp.), is one of the adjuvants accompanying Hp in enhancing the
carcinogenic progress to HGC. The hypothesis is constructed in
two levels: a macroscopic setting for the PtQ flow from bracken
fern to the human diet and contributing factors, and a microscopic
venue describing a sequence of physiochemical events within the
human gastric environment on which the PtQHpHGC edifice
may be construed. To support this idea we examine briefly: (I)
recent inconsistencies in the HpHGC connection, (II) the PtQ
insertion in the diet of people in high risk areas of HGC, and (III)
PtQ carcinogenic chemistry, all of which are instrumental to under-
standing this hypothesis. Further PtQHp combined effects on
proinflammatory reactions and gene disturbance in association
with gastric cancer are propounded as well to bring additional
buttress.
Section (I): does Hp induce GC by itself?
Conventional wisdom has espoused Hp infection with HGC as
the dearth of published reviews and meta-analysis studies expose
[8,2022]. A summary odds ratio of 3.0 (95% CI 2.33.8) for non-
cardia gastric cancer (NCGC) has been estimated for Hp-infected
people [23], leading to NCGC incidence rates attributable to Hp in
the 74.789.0% range [24]. Some obstacles, however, persist,
grouped here in the following seven issues:
(1) This assessment leaves the NCGC etiology of the remaining
11.024.3% unaccounted for, and disregards the potential
contribution of other environmental carcinogens.
(2) While 50 to >90/100 people are infected with Hp in most
places, only one in 2000 Hp-infected people per year,
(0.05/100) ever develops HGC [25].
(3) These figures do not match either the elevated frequency
(4481%) of procarcinogenic Hp cagA+ and vacAs1m+ strains
[20,26,27] despite some HGC/Hp cagA+vacA+ statistical
associations from studies monitoring limited numbers of
subjects [28].
(4) Inconsistencies about NCGC prognosis among people with
and without Hp infection still persist according to a recent
meta-analysis embracing 2454 subjects [29]. Refinement of
data underscores the Hp molecular cascade evolving up to
a precancerous condition but not NCGC itself [22].
(5) Hp eradication programs and HGC outcomes in treated
patients are still controversial [30]. Of the recognized patho-
logical stages induced by Hp infection, all but intestinal
metaplasia can improve after Hp eradication [31]. A decrease
in HGC incidence in the general population following Hp
abolition measures has not been proved up to now.
(6) By contrast, dietary changes have been held causative of the
decline of HGC morbidity in places where prevalence of Hp
continues to be high (Poland: current Hp prevalence, 85
95% among >25 years-old individuals, HGC rates cut in half
between 1960 and 2006 [32]). Hence, diet control rather
than Hp suppression policies seem more valuable to reduce
HGC incidence.
(7) HGC trends differ markedly by geographical location at
country and regional levels [3335,36,37]. At the large scale,
Hp prevalence and GC incidence maps do not overlap but in
a few significant instances [33,38], leading to a number of
geographical enigmas [25,39]. By contrast, other severe gas-
tric diseases elicited by Hp do run in parallel with infectional
rates [39].
The molecular basis of Hp epithelial carcinogenesis is currently
advancing steadfastly but the picture is still incomplete [40,41].
According to some views, Hp may create a gastric environment
conducive to a pre-neoplastic transformation in the mucosa, which
develops further into malignancy upon intervention of other sub-
stances occurring in the complex digestive milieu [14,36].
Section (II): diet in the HGC landscape
Diet is the second most important contributor to HGC [42,43]
and may be responsible for about one third of all cancer cases
among non-smokers. By some estimates, 500010,000 dietary
compounds reach the upper gastrointestinal tract (GI) among
which several have tested positive to mutagenicity tests [44]. Of
special concern is the discovery of an increasing number of geno-
toxic dietary natural products [45,46]. Several studies comprising
large cohorts of participants report on specific diet habits corre-
lated with HGC incidence [6,40,43,47,48] or diet plus Hp [49,50].
Bracken ptaquiloside (PtQ)
PtQ, a terpenoid glucoside, was first discovered in 1983 by
mutagenicity and carcinogenicity-guided chromatographic frac-
tionation of toxic bracken fern (Pteridium aquilinum) [51]. Bracken
is not only highly poisonous to farm animals but PtQ induces a
variety of GI, urinary and mammary malignant tumors in animals
and potentially humans [5256]. PtQ impact is compounded by
other illudanes in bracken and other ferns possessing similar
molecular features [5760].
Capital issues to bracken/PtQ exposure risk
The present hypothesis requires real risk assessment of humans
being exposed to PtQ, a subject generally disregarded by the med-
ical community. A handful of central questions relative to PtQ
ingestion are now highlighted:
(1) Bracken is a most ubiquitous plant and a serious weed pest
of grazing paddocks all over the world except in extreme cli-
mates, exposing animals to feeding on its copious fronds.
Brackens chemical armamentarium is complex and diverse
[54]. In addition to deadly acute toxicosis, farm animals
develop a series of chronic ailments after long exposure to
moderate bracken intake. Some of these syndromes pass
unnoticed at slaughter in many countries [55] since no legal
bracken surveillance regulations are currently in effect [61].
(2) Bracken compounds enter the human diet through eating
bracken fiddleheads and rhizomes. In one well studied
instance in Ouro Preto (central Brazil), consumers of boiled
bracken croziers (Pteridium arachnoideum) had a 5.5 higher
risk of developing HGC than those who never ate this pro-
duct [62]. Substantial residues of PtQ were found in this food
since culinary processing does not destroy but a fraction of
the neoplastic potential [63,64].
 A. Oliveros-Bastidas et al. / Medical Hypotheses 88 (2016) 9199
(3) More importantly, animal products are also a conduit for PtQ
and homologous compounds into the human diet, since
some of these chemicals have been found in animal dietary
materials. For example, ptesculentoside, a close relative of
PtQ from bracken, has been detected in raw meat along with
PtQ (skeletal muscle, 42 lg/100 g) and liver (32 lg/100 g) in
cattle 15 days after feeding a moderate quantity of bracken
(Pteridium esculentum) fronds [65]. There was a three and
fivefold concentration, respectively, in calves feeding
actively on bracken at slaughter time, which illustrates the
slow metabolism of these illudanes. Likewise, PtQ occurs in
milk from healthy cows on a diet containing fresh bracken
[66] in quantities varying from 0.35 to 2.68 mg/100 ml of
unprocessed milk [67] or more [68]. Of major local concern
is the finding of PtQ in milk from an assortment of healthy
farm animals including cow, horse, sheep, goat and donkey,
exposed to circumstantial bracken browsing under normal
animal husbandry conditions in southern Italy [61,69]. Sig-
nificantly, up to 89% of milk samples from cows grazing on
bracken-infested fields tested positive for PtQ in a mountain
zone of Ecuador covering two provinces [68].
(4) As a glycoside of low molecular weight, PtQ is soluble in
water and is exiled from bracken fronds and rhizomes by
rain [70]. Leached PtQ slowly degrades in the ground
depending on temperature, soil type, pH, and microbial
metabolism [71]. PtQ may thus contaminate surface and
well water in the vicinity of bracken swards, albeit in small
concentration [72,73]. From these evidences one may con-
clude that humans are indeed at risk of chronic PtQ intake,
damage in GI and possibly beyond in bracken areas.
Section (III): ptaquiloside chemistry and mode of action
Of special importance to support our hypothesis is the chemical
behavior of PtQ under those conditions found in the human stom-
ach. PtQ chemistry is strongly pH-dependent as the following fea-
tures, portrayed schematically in Fig. 1, illustrate.
Briefly put, PtQ is an amphiphatic compound owing to gluco-
sidic (hydrophilic) and sesquiterpenoidal (lipophilic) residues in
the molecule. This mixed lipophilic/hydrophilic attribute facilitates
dilution in the aqueous gastric and cell cytosol domains and poten-
tially unassisted diffusion across lipid membranes in the upper GI
epithelia. PtQ is unstable in acid or base through pH-dependent
diverging mechanisms A and B (Fig. 1) [74]. While transformation
in aqueous acid (route A) yields innocuous pterosin B (PtB), mild
aqueous base (route B) furnishes strongly electrophilic ptaquilo-
dienone, also known as active ptaquiloside (PtA). PtA, a key inter-
B A
Ptaquiloside
slightly alkaline
(PTQ)
aqueous medium Glucose Glucose
acid catalysis
Activated PTQ Pterosin B
(PTA) (PTB)
DNA Nucleoside
Gene silencing
DNA-PTA Gene malfunction
adduct
Oncogene expression
DNA fragmentation
& repair
Fig. 1. pH-dependent conversion tree of ptaquiloside (PtQ), the main carcinogen of
bracken fern, to intermediates of enhanced bioactivity, particularly activated
ptaquiloside (PtA) and terminal products including innocuous pterosin B (PtB) and
DNA-adducts furnishing gene transmogrification.
93
mediate for carcinogenesis, is chemically reactive, but it can
nevertheless survive for limited periods of time under specific
physiological conditions [59]. This compound may be further con-
verted to PTB, form adducts with free amino acids and proteins, or,
more importantly, undergo chemical insertion onto DNA bases as
alkylating agent [75].
DNA alkylation by PtQ/PtA causes DNA depurination, transgres-
sion, and double strand breakage [76], a hallmark of cellular neo-
plastic transformation [3]. From there on, an array of genotoxic
activities take place in a dose-dependent fashion and low concen-
tration (5 lg/ml) [77]. PtQ genotoxicity of gastric cells is expressed
as deregulation of several genes related to DNA damage signaling
cascades and repair, as reported for living mice [78]. Likewise,
cytotoxicity of PtQ on human HGC-27 gastric epithelial cell line
at 2.56 lg/ml is also on record [79]. Both phenomena are likely
associated with the initiation of carcinogenesis.
On the other hand, the contribution of route B is negligible at
pH < 5.0, but becomes dominant at pH 6.39 0.28 and above
(Fig. 2) [80]. This pH threshold, here named the Ayala limit to
acknowledge its source, is of paramount importance to our model
(see next section). All other bracken illudanes obey the reactions of
Fig. 1 under identical conditions [86] and are, therefore, potential
carcinogens.
Conversion of PtQ to PtA under physiological conditions
Because PtA is the most active carcinogenic form of bracken PtQ
[81], our hypothesis needs to account for physiological conditions
modified by Hp which prompt the conversion of PtQ to PtA within
the constraints of the Ayala limit.
There are two settings in the GI medium to bring about this
reaction: (1) Loss of glucose from PtQ by a pancreatic glucosidase
and (2) a medium of pH above the Ayala limit to promote route
B. The first of these reactions would not impinge on NCGC as it
takes place in the transpyloric domain. As regards to the second
option, few organs provide a medium alkaline enough to evoke
the PtQ to PtA transformation. Ileum and urinary bladder in the
rat model provide such conditions, precisely where malignant
tumors can be induced in 100% of ACI rats after chronic oral
administration of small amounts of PtQ (0.0270.08% of diet)
[52]. Additionally, PtA-DNA adducts have been detected in the
ileum of rats and calves fed bracken foliage [64,82]. Similarly, uri-
nary bladder in the cow (pH 7.68.2) develops cancerous lesions
upon PtQ oral uptake. This condition is a well characterized disease
known as bovine enzootic hematuria (BEH) [55,83]. BEH is also a
Mixed mechanism zone
Route A Route B
0
zone zone
aqueous acid
medium
-1
log Kobs
(innactive)
-2
-3
TUMORIGENESIS
-4
2 3 4 5 6 7 8 9 10
pH
Fig. 2. Semi-log plot of observed decomposition rates (log Kobs) of ptaquiloside as a
function of pH in water at room temperature, showing different molecular
outcomes. Routes A and B refer to mechanistic pathways described in Fig. 1.
Redrawn from Ayala-Luis et al. [80] with permission. ( John Wiley & Sons, 2006,
license N 3630130760725).
94 A. Oliveros-Bastidas et al. / Medical Hypotheses 88 (2016) 9199
key component of the first macroscopic model of our hypothesis,
which is now described.
A macroscopic PtQHpHGC model
Based on this background the PtQ molecular framework finds a
potential epidemiologic association with HGC. In designing a con-
necting construction we superimpose four elements in one geo-
graphic unit: brackenanimalhumanHp. This crossroads is
further defined by three convergent, although independent, lines
of evidence:
(1) Animals: BEH reaches epidemic proportions in extended
bracken-infested mountainous zones along the Central
American range and the Andean sierras of South America,
precisely where HGC rates are highest and NCGC constitute
75100% of HGC cases. HGC rates are much lower in the low-
lands where bracken ferns rarely grow [34,37,8486]. Signif-
icantly, Chile, a bracken-free country, does not follow this
trend, showing statistically undifferentiated HGC rates in
high and lowlands of comparable latitude [38]. Social deter-
minants constitute the main HGC risk factors there [87].
(2) Humans: the bracken-HGC association was first exposed in
Wales in 1990 with an estimated HGC relative risk of 2.34
for people living in rural bracken areas during childhood
and consuming buttermilk, a likely PtQ vehicle, as compared
with individuals from rural non-bracken regions [88]. Simi-
lar conclusions were drawn from regional studies in Costa
Rica [84] and the Venezuelan Andes [34], including HGC
clusters of unusually large mortality rates there [85,89].
(3) Hp prevalence is high (>85% and up to 96%) among peasants
in cattle homesteads under deprivation, low education, defi-
cient sanitation, menial jobs and crowding [90].
The emerging model contemplates PtQ transfer from plant to
people through three branches: (1) fern croziers used as food; (2)
meat products and milk from animals consuming bracken with
attendant damage to cattle via acute bracken poisoning (ABP)
and bovine enzootic hematuria (BEH); and (3) drinking water from
bracken sward runoff, wells or downhill springs. While the contri-
bution of the latter is small, drinking bracken runoff water conceiv-
ably maintains long term stomach exposure to PtQ. Additional HGC
enhancers include host lifestyle (diet choices and abundance) and
individual predisposal owing to proinflammatory genetic polymor-
phism [91]. This is particularly notorious among people carrying
the proinflammatory interleukin IL1RN*2 variant (OR 3.1, 95% CI
1.56.5) [6,92,93].
Pursuant to legitimate part of this model, Peraza and co-
workers [85] confirmed the co-occurrence of HEB, medium to high
density bracken swards and extremely high HGC death rates (82.2
103.6 per 100 K people) in specific districts of Tchira State, in the
Venezuelan Andes. This area is in close geographical vicinity to the
HGC cluster found by us [89] with similar phytogeographical fea-
tures, and in consonance with observations in Costa Rica [84]. This
model applies to small communities and townships strongly
dependent on local food production where animal products are a
basic staple and not to the general population of large urban areas
due to the high dilution of bracken toxins in pooled dairy and meat
products.
PtQHp combined carcinogenesis, a microscopic model
The macroscopic portrayal of the model above includes Hp as a
cofactor since there is a range of conceivable PtQ/Hp manners of
interaction. These are condensed in our five-stage hypothetical
model (Fig. 3) which focuses on: (A) gastric luminal medium; (B)
gastric mucus structure and mucin degradation elicited by Hp;
(C) bacterial pH gradient modification of the gastric mucosa that
favors PtQ survival and penetration into epithelial tissue; (D) com-
bined PtQ/Hp effects on gastric immune and inflammatory
responses; (E) PtQHp complementary activity at selected cell sig-
naling cascades and genome disturbance. A description of this por-
trait follows.
(A). Hp, PtQ and the gastric luminal medium
Mid gastric and antral pH is generally low in healthy people,
with fluctuations in the 1.32.8 range for most of the 24 h period.
This is a hostile environment for PtQ integrity prompting its
decomposition to innocuous pterosin B (PTB) via route A
(Fig. 1). As a result, PtQ should not constitute a particularly high
risk HGC effector in a healthy adult stomach. Three circum-
stances, however, may elevate the antral pH to near neutrality
or above:
(1) Cyclic postprandial episodes of higher pH reaching up to 5.0
due to food and gastric juice mixing-dilution. Even lower
proton concentrations are achieved during treatment with
widely used H+/K+-ATPase drug inhibitors such as pantopra-
zol [94]. Certain popular foods like milk, a recognized vehicle
of PtQ as said above, possess a momentary neutralizing buf-
fer effect on gastric acid [95].
(2) Hypochlorhydria and achlorhydria driven by gastric atrophy
as a result of advanced Hp infection [96].
(3) Transpyloric duodenogastric reflux (DGR). These episodes
drive antral pH to 7.0 and above beyond the pH limit of
the A to B route transition, the Ayala limit (Fig. 2). DGR
bursts may occur several times in the same day, particularly
during supine time [97] and may last for several minutes.
DGR is exacerbated in Hp patients, and subsides significantly
after eradication therapy [98]. This event should allow PtQ in
the diet to maintain its molecular integrity and diffuse safely
to the gastric mucus layer and eventually to the epithelial
surface aided by Hp colonization strategies (see stage B).
Importantly, DGR is also an independent risk factor for antral
intestinal metaplasia in people or gastric adenocarcinoma
(rat), Hp status notwithstanding [99,100]. Oxidative stress
via COX-2 signaling pathway has been suggested as a con-
tributing factor [101].
(B). PtQ, H. pylori-induced mucin pH modulation and degradation
The gastric mucosa is protected from ingested noxious agents,
bacterial infection and the strongly acidic medium of the digestive
lumen by a 300 lm layer of viscous mucus and the glycocalyx
mucus coat just beneath it, in close contact with the luminal side
of epithelial cell microenvironment [102]. Both layers are consti-
tuted by tightly adherent and heavily glycosylated proteins:
mucins. To colonize the mucus niche, newly arrived Hp must swim
across this viscous barrier to reach eventually the epithelial sur-
face. In fact, most of the Hp bacterial load (80%) lives in this mucus
forming an infectious pool but without causing gastric disease
[103]. Only a small portion of the bacterial population ever gains
access and adheres to the epithelial surface, pathologically sub-
verting cell function. Ammonia produced by Hp urease utilizing
efficiently urea in the mucus as substrate and additional urea
intake from the impulse of urel, a Hp transmembrane urea trans-
porter protein, raises the pH of the bacterial microenvironment
from 2 to 6 and beyond [104]. Conversion of the equimolar amount
of CO2 produced by the urease reaction to bicarbonate by a-
carbonic anhydrase, a Hp periplasmic enzyme, also contributes to
alkalinization [105,106] providing a buffering effect. Epithelial
 A. Oliveros-Bastidas et al. / Medical Hypotheses 88 (2016) 9199 95

Fig. 3. Microscopic model of the ptaquiloside-Helicobacter pylori interaction during penetration of the mucus layers from gastric lumen to the epithelial cytoplasm domain.
PtQ = Ptaquiloside; PtA = ptaquilodienone or activated ptaquiloside; PtB = Pterosin B; Hp = H. pylori bacterium; Ad = Cell adhesins; Nu = Cell nucleus showing DNA strand.
DGR = Duodenogastric reflux. Stages: (1) and (2): PtQ ? PtB uneventful conversion in gastric bolus and unmodified mucus at low pH. (3) and (4): Hp creates higher pH
microenvironment allowing PtQ survival, which (5) is translocated to the cytoplasm domain; (6) PtQ ? PtA conversion (route B) occurs in >pH 7.1 medium furnishing PtA-
DNA adducts and damage; (7) CagA/VacA Hp toxins in cytoplasm disrupt cell-to-cell adherence (8) allowing PtQ/PtA to penetrate further (9) into underlying cell layers; (10)
DGR pulses raise pH to 7.0 and above, enhancing the PtQ ? PtA conversion; (11) Mucus layer and acid secretions are reduced at atrophic gastritis stage furthering PtQ/PtA
infiltration.

damage stemming from Hp insult enhances bicarbonate secretion
as well [107].
While it was originally thought that Hp had no effect on mucin
viscosity [108], more recent investigations gave credit to a dra-
matic increase in mucus viscoelasticity after Hp invasion [109].
Mucin structure is also weakened by the breakage of disulfide
bonds elicited by a thioredoxin system in Hp [110]. The combined
effects turn the mucin gel into a more fluid substance that facili-
tates the rapid movement of Hp in the mucus [109]. Diffusion of
water-soluble compounds in the lumen such as bracken carcino-
gen glycosides may thus be fostered concurrently by Hp and in pro-
portion to bacterial offense.
An additional mechanism of PtQHp cooperation with regard to
gastric mucus consists of the inhibition by Hp of mucin synthesis in
epithelial cells while decreasing substantially the rate of mucus
replacement in the gastric mucosa [111]. By frequent mucus
renewal the stomach clears bacteria and noxious alimentary com-
pounds that may invade, diffuse or dissolve there. A slower mucin
turnover rate not only creates a more viable ecosystem for Hp sur-
vival but also for PtQ molecular persistence.
Taken together, Hp escorts PtQ to the epithelial surface as a
result of modification of the surrounding microenvironment favor-
able to both entities. As infection progresses, mucus pH and elas-
ticity become increasingly mild for PtQ integrity and diffusion
toward epithelial cells.
(C). pH of the gastric epithelial surface and cell cytoplasm
Once Hp adheres to the epithelial cell membrane, key patho-
genic factors are translocated from the bacterium to the cytoplasm
domain, severely altering its biochemistry. One of these factors is
vacuolating cytotoxin VacA produced by vacA + Hp genotypes. Of
interest to our hypothesis, VacA toxin enhances efflux of bicarbon-
ate from epithelial cells by 50% at only 40 nM concentration after
140 min exposure, as a result of the formation of anion-specific
pores in the cell membrane [112]. A significant drop in proton
secretion occurs simultaneously, with an expected overall pH ele-
vation of the glycocalix. Authors Debellis and coworkers [112]
employed a sophisticated microelectrode technique for measuring
the intracellular pH of surface epithelial cells. It was stable at 7.4
under normal conditions but underwent slight acidification to 7.1
after luminal exposure to 40 nM VacA toxin because of bicarbonate
efflux. Both values fall beyond the Ayala limit, hence any PtQ dif-
fusing into the epithelial glycocalix and cell cytoplasm would be
converted to PtA by route B of Fig. 1. Once inside the cell, PtA
would be translocated to the nuclear domain to alkylate DNA lead-
ing to the annotated procarcinogenic damage. As an additional fac-
tor in our model, VacA also debilitates intercell adhesion with loss
of epithelial structure, creating openings in the cell layer to further
PtQ/PtA intraepithelial penetration.
The PtQ/Hp collaborative sequence of events so far described is
illustrated schematically in the eleven stages of Fig. 3. As the
mucus layer recedes with atrophic gastritis progress, the PtQ
lumen-to-epithelium transfer is facilitated. Additional mutual con-
tributions to HGC can be proposed or are the result of recent exper-
imental evidence. These are condensed in sections D and E.
(D). PtQ, Hp, immune and inflammatory responses
PtQ poisoning in cattle is characterized by acute leucopenia
generated by severe bone marrow aplasia. Immunomodulatory
effects of PtQ involve monocytes, dendritic cells, and natural killer
cells (NK) with an overall diminished host immune response to
infectious disease and antitumor immunosurveillance [113,114].
96 A. Oliveros-Bastidas et al. / Medical Hypotheses 88 (2016) 9199
In humans, low NK activity is associated with a greater risk of can-
cer, [115]. Consequently, PtQ-elicited immunodepression collabo-
rates with Hps own strategy to evade the host innate immune
and adaptive responses as well as to thwart cancer progression
[116].
On the other hand, inflammation is a complex process involving
multiple cytokines and protein effectors frequently associated with
cell signaling cascades possessing a succession of branched and
intertwined pathways. This scenario raises the possibility of addi-
tive or synergistic proinflammatory activity of different inducers
acting on various cell membrane receptors and downstream sig-
naling cascades. A specific case of PtQHp interaction in this regard
has appeared recently [117]. Mice with and without Hp infection
were given a bracken water extract (BWE) containing PtQ. Unin-
fected mice provided with BWE developed mild gastric inflamma-
tion, whereas Hp mice with no BWE showed mild to moderate
inflammation. By contrast, Hp + BWE mice developed severe
inflammation in the same time period. An obvious HpPtQ interac-
tion leading to important pathogenic changes in the gastric mucosa
had occurred, possibly through the sequence of Fig. 3, but the
molecular mechanism remains to be elucidated.
(E). PtQ/PtA and Hp complementary gene alterations in gastric mucosa
Both Hp and PtQ/PtA share the capacity to induce DNA instabil-
ity of gastric cells as well as DNA double strand breaks (DSB)
[78,118]. Transcriptome analysis of natural killer cells (NK)
exposed to PtQ showed that 872 genes were affected, 77 of which
were up-regulated and 795 were down-regulated [114]. Upon oral
administration of PtQ to mice, a panel of genes related to DNA
damage signaling pathways and repair were found to be equally
deregulated in gastric epithelial cells [78]. Of the set of oncogenes
modulated by Hp, H-ras p21 appears prominently overexpressed
[119], a frequent response in HGC and other cancers [120]. Ras pro-
teins are fundamental components of cell signaling pathways asso-
ciated with cell proliferation, differentiation and survival. Mutation
of this gene is believed to occur during advanced Hp pathology
[119] but actual induction by Hp has never been proved. Nor has
any mutation in tumor suppressor gene p53 ever been recorded
from Hp activity [16].
As regards to PtA, an abnormal H-ras protein structure results
from PtA alkylation of an adenine residue in codon 61 of the H-
ras gene. This PtA-DNA alkylation leads to an A to G transition or
A to T/C transversion at specific codon sequences, which upon
transcription and synthesis yields a defective Ras-GTP protein
complex [82]. This abnormal complex is unable to switch off the
H-ras signal cascade furnishing loss of control in cell proliferation
and survival. Silent H-ras mutations and overexpression have also
been observed in the urothelium of bracken exposed cows [121].
As well as, gastric mucosal cell proliferation occurs with atten-
dant tumor suppressor p53 gene overexpression and/or point
mutations [122]. Gene p53 encodes a transcription factor critically
related to DNA repair and apoptosis and is found in HGC, fre-
quently in mutant form [123]. In vitro human gastric cells and
in vivo animal studies show that exposure to PtQ/PtA does in fact
cause somatic mutations in these two genes [78,81,82,124] but
not Hp [125]. Moreover, expression of p53 is enhanced whereas
several genes associated with DNA damage signaling cascades
and repair are deregulated under the influence of purified PtQ
[78]. Therefore, the PtQHp combined attack on gastric epithelial
cells constitutes a highly potentiated pro-malignancy disturbance
at the oncogene level.
Finally, another recently unveiled manner of PtQHp interaction
[117] consists of a set of histomorphologic and transcriptional
alterations in the expression of several genes in the gastric mucosa
related to Hp anchoring to the epithelial cell membrane. Prominent
among the bracken water extract (BWE) effects fed to mice was
over-expression of epithelial cell gene B3galt1 encoding the syn-
thesis of galactosyltransferases. These enzymes participate in the
extension of mucin oligosaccharide chains during mucin-1 cell-
to-glycocalix recycling, while Fut4 encodes a fucosyltransferase
which is pivotal for chain extension of terminal Lewis and fucosy-
lated structures linked to Sialyl LewisX antigen caps. Within the
epithelial microenvironment of the glycocalix, these structures
are instrumental for the binding of Hp to the epithelial cell surface
through membrane sialic acid-binding adhesins, BabA and SabA,
favoring Hp establishment [126]. While Hp alone enhanced the
expression of Sialyl LewisX antigen as much as BWE by itself in
mice, the response was much stronger in the Hp infected mice
under the BWE diet. A cooperative link of BWE and Hp at the bac-
terial encroachment level was thus demonstrated experimentally
[117]. The hypothetic model of HpPtQ collaboration in HGC
shown in Fig. 3 seems, therefore, well supported. Other dietary car-
cinogens may find similar penetration pathways.
Concluding remarks
Though there is epidemiological and experimental evidence
linking bracken toxins to animal and human carcinogenesis, as
well as H. pylori (Hp) infection with gastric neoplastic progress,
no mechanistic association between these two oncogenic entities
has ever been proposed save for a recent demonstration of the
improved adherence of Hp to epithelial cells elicited by bracken
aqueous extracts [117] and collaborative proinflammatory reaction
in the gastric environment. The set of hypotheses here proposed to
strengthen mechanistically this bracken illudane-Hp association
may help in future experimental design and perhaps in conceiving
new bracken/Hp eradication policies aimed at reducing substan-
tially the gastric cancer rates and animal economic losses brought
about by this pernicious fern-weed. Similarly this hypothesis
might be expanded to other carcinogens occurring in human food
and beverages.
Conflict of interest statement
Authors declare no conflict of interest.
Acknowledgements
Authors are grateful to the ADG-GQE and Consejo de Desarrollo
Cientfico, Humanstico, Tecnolgico y de las Artes, Universidad de
Los Andes Venezuela funds for supporting bracken and H. pylori
research in our group that encouraged this hypothesis, and logistic
support of Universidad de Valencia Library Services in Spain.
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