Creatine and Creatinine Metabolism - Physiological Reviews

July 2000 CREATINE AND CREATININE METABOLISM 1165
first reaction is N-hydroxylation of the exocyclic amino
group, yielding the respective N-hydroxy-AIA. For most
AIA, this reaction is catalyzed predominantly by the cytochrome
P-4501A2 isoenzyme (296, 330, 872, 920). H 2O 2 in
the physiological/pathological concentration range considerably
stimulates metabolic activation of IQ by human
and rat cytochrome P-4501A2 (19). PhIP, on the other
hand, is hydroxylated at similar rates by cytochromes
P-4501A1 and 1A2 (212, 330). In addition, a series of other
cytochrome P-450 isoenzymes (see Refs. 158, 235, 345,
590, 872) as well as prostaglandin H synthase (670) were
suggested to participate in N-hydroxylation of AIA, but
their actual contribution to the metabolism of the respective
AIA is only vaguely known.
N-OH-AIA can be activated further by O-acetylation,
O-sulfonylation, O-prolylation, or O-phosphorylation (see
Refs. 119, 159, 235, 743, 872, 920). In human liver, Oacetylation
is mainly catalyzed by the polymorphic Nacetyltransferase
(NAT2) isoenzyme and yields the respective
N-acetoxy-AIA (345, 1121). In human mammary
gland, on the other hand, O-acetylation is catalyzed predominantly
if not exclusively by the NAT1 isoenzyme
(828). O-Sulfonylation of N-OH-PhIP is catalyzed in human
liver by the thermostable phenol sulfotransferase
isoenzyme (TS-PST), whereas the contributions of thermolabile
phenol sulfotransferase (TL-PST) and dehydroepiandrosterone
sulfotransferase (DHEA-ST) seem to be
negligible (126). Moreover, no O-sulfonylation was observed
for N-OH-IQ and N-OH-8-MeIQx in human liver.
While O-prolylation of N-OH-AIA is catalyzed by L-prolyltRNA
synthetase, the enzyme responsible for ATP-dependent
O-phosphorylation has not yet been identified. All
four activated AIA metabolites are likely to decompose to
the highly reactive nitrenium ion (872; see also Ref. 348)
which then reacts with protein, low-M r thiols like glutathione
or cysteine, or DNA.
Each AIA forms several different adducts with DNA
(e.g., Ref. 110), but most of them involve guanine bases.
So far, IQ, MeIQ, 8-MeIQx, 4,8-DiMeIQx, and PhIP were
reported to form adducts with 2�-deoxyguanosine, by
binding of the exocyclic nitrogen of the AIA to the C-8 of
guanine (see Ref.s 235, 617, 872, 886, 922). To a lesser
extent, IQ and 8-MeIQx also formed adducts with the N 2
of guanine. The preference of activated AIA for guanine
bases is in perfect agreement with the fact that most
mutations induced by IQ, MeIQ, 8-MeIQx, and PhIP in the
p53, Ha-ras, Ki-ras, APC, supF, lacZ, lacI, and hprt genes
involve G-C base pairs (see Refs. 296, 682, 872, 1013).
In addition to these activation reactions, several detoxification
pathways have to be considered, in particular
N-glucuronidation, N 2 -sulfamation, ring hydroxylation
followed by sulfation or glucuronidation, N-demethylation,
and glutathione- plus GST-mediated suppression of
AIA-DNA adduct formation (see Refs. 215, 235, 448, 521).
N-Glucuronidation has been established for N-OH-IQ,
N-OH-8-MeIQx, and N-OH-PhIP. In the case of N-OH-PhIP,
two distinct glucuronides, namely, N-OH-PhIP N 2 -glucuronide
and N-OH-PhIP N 3 -glucuronide, were identified,
with their ratio differing considerably between rat, dog,
and human. Ring hydroxylation, like N-hydroxylation, is
catalyzed by cytochrome P-4501A isoenzymes, although
different isoenzymes may be involved in the two processes.
In the case of PhIP, ring hydroxylation is catalyzed
predominantly by cytochrome P-4501A1 and yields 4�-OH-
PhIP, which displays no or only low mutagenicity. In
addition, 5-OH-PhIP may be formed via a cytochrome
P-450-independent pathway.
In in vitro studies, glutathione inhibited the covalent
binding of N-acetoxy-IQ and N-acetoxy-8-MeIQx to DNA
by �40%, and further addition of human or rat GST isoenzymes
had no extra effect (561). In contrast, covalent
DNA binding of N-acetoxy-PhIP was only slightly inhibited
by glutathione alone, but inhibition was greatly amplified
by further addition of human GST isoenzyme A1–1
or rat GST isoenzyme 1–2. The human GST isoenzymes
A1–2 and P1–1 as well as rat GST 12–12 had lesser effects.
Analysis of incubation mixtures containing N-acetoxy-
PhIP, reduced glutathione, and GST A1–1 failed to reveal
glutathione conjugates of PhIP. Only oxidized glutathione
and PhIP, in a molar ratio of �1:2, were detected as
reaction products.
The contribution of the different activation and detoxification
reactions to the total metabolism of a given
AIA differs to a large extent between individual AIA,
between different tissues, and between species. It therefore
goes without saying that our current knowledge on
this matter is still incomplete. In humans, after the consumption
of a test meal, 90.8 and 69.9% of 8-MeIQx and
PhIP, respectively, were metabolized via cytochrome
P-4501A2, 2.6 and 1.2% were excreted unchanged in the
urine, and 6.6 and 28.9% were eliminated by other routes
of metabolism (81). Metabolic activation of AIA by Nhydroxylation
and O-acetylation occurs predominantly in
the liver of rodents and primates. Accordingly, for all AIA
except PhIP, highest DNA adduct levels are found in liver,
followed by kidney, heart, and colon (160, 920). For PhIP,
on the other hand, highest DNA adduct levels were detected
in rat pancreas, followed by prostate, colon, lung,
and heart, whereas liver contained 25- to 50-fold lower
levels of PhIP-DNA adducts than the pancreas (447, 886).
It is surprising that metabolic activation of PhIP nevertheless
occurs predominantly in the liver and that Nhydroxy-PhIP
and N-acetoxy-PhIP are the major metabolites
that subsequently are transported via the blood
circulation to those extrahepatic tissues that are subject
to the mutagenic effects of PhIP (see Ref. 872). This
apparent discrepancy suggests that an efficient detoxification
mechanism is operative in the liver. Evidence supporting
this notion in fact exists, indicating that protection
may be brought about by glutathione and GST (539,