1426

M. B. ABOU-DONIA ET AL.
alkaloids (vinblastine and vincristine); i.e., increased P-gpefflux lowers intracellular drug
concentrations in neoplastictissues (Gottesman et al., 2002). CYP3A4 and CYP2D6(human
analog of rat CYP2D1) are involved in the metabolismof a vast number of prescription
medications (Rendic &DiCarlo, 1997; Michalets, 1998; Clarke & Jones, 2002), andincreased
expression of these CYP isozymes results in accelerateddrug metabolism and hence reduced
clinical efficacy.The present finding of increased expression of P-gp andCYP proteins by
Splenda at the low dosages used in thisexperiment is clinically important with regard to potential
druginteractions. For example, it is known that P-gp becomes satu-rated when large doses of
certain drugs are administered (Lin& Yamazaki, 2003). Splenda-enhanced expression of
P-gp,however, makes saturation unlikely even in the presence of high doses of drugs. That is, if
the results in rats are similar forhumans, the magnitude of the elevation of P-gp consequent
toconsuming Splenda over time might lead to extrusion of highdoses of drugs. Furthermore, in
cases of drugs such as digoxinthat are given at a low oral dose (0.25–1 mg), it is unlikely
thatP-gp becomes saturated even at basal level.While the increase in the expression of CYP3A4
andCYP2D1 was linear and dose dependent, the expression of P-gp was nonlinear. For P-gp,
there was a 2.43-fold increaseat 300 mg/kg/d Splenda (i.e., 3.3 mg/kg/d sucralose) and
a2.23-fold increase at 500 mg/kg/d Splenda (i.e., 5.5 mg/kg/dsucralose) followed by a
precipitous suppression of P-gprelative to control at the 1000 mg/kg/d Splenda dose
(i.e.,11mg/kg/d sucralose). A possible explanation for the significantreduction (not increase) of
P-gp expression at the 1000 mg/kgSplenda dose may be due to a marked increase in
CYPisozyme expression at the highest dose, i.e., 2.51-fold forCYP3A4 and 3.49-fold for
CYP2D1 expression. Thisenhanced expression of CYP isozymes likely metabolizessucralose and
decreases its bioavailability so that it no longerexists intact in the small intestine at levels
sufficient toincrease P-gp. That is, the effective dose of sucralose at thehighest dosage of Splenda
would actually be far lower at theintestinal level than the 11 mg/kg/d of sucralose
consumedorally because it is degraded by CYP3A4 and/or CYP2D1.This explanation is
supported by the observation that P-gp andCYP3A4 have common tissue distribution and are
known tointeract in a coordinated manner in enterocytes (Mathenyetal., 2001; Eagling et al.,
1999; Wacher et al., 1998). Theeffects on P-gp and CYP enzymes seen here cannot be due tothe
maltodextrin component of Splenda because it is hydrolyzed
TABLE 3
Summary of the Effects of Splenda Observed at the End of the 12-wk Treatment Period and at
the End of the 12-wk RecoveryPeriod (No Splenda), With Sucralose Level Contained in the
Splenda Product Given as WellEffectsDosage of Splenda (mg/kg/d)Sucralose (mg/kg/d)
a
After 12-wk treatmentAfter 12-wk recovery1001.1Decrease of beneficial intestinalbacteria;
increased fecal pH;increased body weightTotal anaerobic bacteria remainedsuppressed; body
weightremained elevated3003.3Decrease of beneficial intestinalbacteria; increased fecal
pH;histopathological changes in thegut
b
; increased P-gp, CYP3A4,and CYP2D1Total anaerobes andbifidobacteria remainedsuppressed;
fecal pH remainedelevated; P-gp remainedslightly elevated5005.5Decrease of beneficial
intestinalbacteria; increased fecal pH;histopathological changes in thegut; increased P-gp,
CYP3A4,and CYP2D1Total anaerobes andbifidobacteria remainedsuppressed; fecal pH
remainedelevated; body weightincreased; P-gp and CYP2D1remained elevated100011Decrease
of beneficial intestinalbacteria; increased fecal pH;histopathological changes in thegut; decreased
P-gp, increasedCYP3A4 and CYP2D1Total anaerobes remainedsuppressed; fecal pH
remainedelevated; P-gp reboundedbeyond control; CYP3A4 andCYP2D1 remained elevated
a
The sucralose concentration in the Splenda product utilized was 1.1%.
b
Lymphocytic infiltrates in the epithelium not seen in controls.
D o w nl o ad ed B y : [ J e a n n e t t e S a n ti n o , Ph D] A t : 19 :30 1 O c t ob e r 2008

EFFECTS OF SPLENDA IN MALE RATS

1427in the duodenum to glucose and then rapidly absorbed (Engferet al., 2000; Booth, 1994). In
contrast, the sucralose portion of Splenda, for the most part, stays in the gut (
Federal Register
,1998).Our results show that the Splenda-induced increase inCYP450 proteins was linear and did
not level off throughoutthe dosage range used here that was equivalent to 1.1–11
mg/kgsucralose. It is likely that higher doses of sucralose saturateCYP isozymes resulting in
decreased metabolic activity of these enzymes. Prior studies examined the metabolic profile of
sucralose in rats treated with chronic, nonphysiological “mega”doses (e.g., 2.2 g/kg/d sucralose)
(Sims et al., 2000; Mann etal.,2000). This mega-dosage in rats is equivalent to 20 g/kg/d of
Splenda or 1.4 kg/d for a 70-kg human. Rats given a diet con-taining a mega-dosage of sucralose
for 18 mo reportedlyexcreted sucralose unchanged in the feces without metabolism(Sims et al.,
2000). This high dose of sucralose may have satu-rated the CYP metabolism enzymes, thus
impeding the body’sability to metabolize sucralose. As a result, P-gp and/or otherABC
transporters played a quantitatively significant role in theextrusion of sucralose and its
subsequent excretion from thebody, mostly in the feces as the parent compound. P-gp
wouldcontinue to be induced at elevated sucralose concentrations if CYP is not removing
sucralose from the GIT.The impact of Splenda (and particularly the chlorocarbonsucralose) on
both P-gp and CYP is consistent with previousreports that chlorinated hydrocarbons interact with
efflux trans-porters and cytochrome P-450 isozymes (Lanning et al., 1996;Bois et al., 1998; Bain
& LeBlanc, 1996; Leslie et al., 2005; Poetetal., 2003). The increased expression of P-gp and
CYP3A4 mayinvolve induction of gene expression via the nuclear pregnane Xreceptor (PXR).
Several lines of evidence indicate that PXR regu-lates gene expression of CYP3A4 and other
P-450 isozymes aswell as the
MDR1
gene that encodes P-gp (Tompkins & Wallace,2007; Geick et al., 2001). Furthermore, the PXR
receptor wasshown to be activated by organochlorine compounds (Kliewer,2003; Medina-Diaz
et al., 2007; Jacobs et al., 2005). The enhancedexpression of P-gp and CYP isozymes found in
this study mayexplain why 65–95% of the sucralose administered orally is report-edly not
absorbed from the gastrointestinal tract (
Federal Register
,1998). Data presented suggest that sucralose undergoes two pro-cesses during first-pass
metabolism in the intestinal membranes:(1) P-gp extrudes sucralose from the intestinal walls
back to thelumen and (2)CYP enzymes metabolize the compound. Theseresults explain why
sucralose, though lipid soluble (Miller, 1991;Wallis, 1993; Yatka et al., 1992), was reported to be
poorlyabsorbed from the gastrointestinal tract. Overall, the elevatedexpression of P-gp and CYP
explains the low bioavailability of sucralose (
Federal Register
, 1998); i.e., it invokes the same presys-temic first-pass metabolism mechanisms as drugs and
toxicants.In conclusion, the findings of this study indicate thatSplenda suppresses beneficial
bacteria and directly affects theexpression of the transporter P-gp and cytochrome
P-450isozymes that are known to interfere with the bioavailability of drugs and nutrients.
Furthermore, these effects occur atSplenda doses that contain sucralose levels that are
approvedby the FDA for use in the food supply.
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