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PERGAMON Archives of Oral Biology 43 (1998) 629±632

Discoloration of dental carious lesions (a review)

G.A. Kleter
Department of Cariology Endodontology Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA), Louwesweg 1, 1066
EA, Amsterdam, The Netherlands
Accepted 7 April 1998

Abstract

The discoloration of dental carious lesions is a marked feature which has received relatively little attention from
dental researchers. In this short review, possible causes are considered: the formation of Maillard pigments,
melanins, and lipofuscins, and the uptake of food dyes, metals, and bacterial pigments. It is concluded that the
Maillard reaction between proteins and small aldehydes produced by bacteria probably accounts for the
discoloration. # 1998 Elsevier Science Ltd. All rights reserved.

Keywords: Caries; Pigments; Review

Dental caries is generally acknowledged as a process
whereby bacterial acids destroy hard dental tissues. It
is therefore not surprising that a great deal of caries
research has been devoted to the de- and remineraliza-
tion of enamel or dentine. Another notable but less
investigated feature of the caries process is lesion dis-
coloration. In the course of the caries process, various
consecutive stages can be recognized by their colour in
enamel: initial lesions appear as opaque white spots
and arrested lesions as brown spots (Ripa, 1977). In
root-surface caries, the generally accepted view is that
an incipient lesion is slightly brown and becomes dark
and hard on probing after caries arrest (Banting, 1991;
Fejerskov and Nyvad, 1986). One recent report, how-
ever, describes soft, black, active lesions (Lynch and
Beighton, 1994). In the super®cial white-spot enamel
lesion, light is di€racted di€erently from that in the
surrounding sound mineral, which causes the chalky
white appearance. Such a physical phenomenon, how-
ever, cannot account for the brownish appearance of
root dentine in incipient lesions and the dark brown
and black colours of more advanced lesions in enamel
and dentine lesions.
There are some publications dealing speci®cally with
tooth discoloration during caries, most of which date
back to the 1950s and 1960s, and have been reviewed
by Van Reenen (1955) and Armstrong (1964). A num-
ber of di€erent hypotheses have been brought forward,
but the supporting evidence does not meet present
standards. In many cases, the investigators assumed
that the discoloration was caused by reactions invol-
ving amino acids released from the dental matrix by
proteolysis. Proteolysis of dental matrix has a key role
in cavity formation according to the proteolysis±chela-
tion theory. As the opposing acidogenesis theory
gained increasing support in the 1960s, scientists prob-
ably lost interest in both proteolysis±chelation and
reactions of amino acids.
Investigators often simulated browning reactions on
dental tissues or pure biochemical compounds under
rather unnatural conditions in vitro. The resemblance
between in vitro and in vivo gross features, such as el-
emental composition, colour, and collagen degradabil-
ity, was inferred as proof that the same reaction would
occur in vivo. For example, Reiss (1938) showed that
isolated cariogenic bacteria induced browning of pro-
tein in the presence of the phenolic amino acid tyro-
sine, similar to melanin formation. Melanins are
pigments that occur in hair and skin and are formed
by the oxidation of tyrosine. Melanin formation is
sometimes referred to as enzymatic browning.
Dreizen, Armstrong and their coworkers focused on
the reaction between sugar and either proteins or
amino acids. Most of their work dealt with the colour,

0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 0 3 - 9 9 6 9 ( 9 8 ) 0 0 0 4 8 - X
630 G. A. Kleter / Archives of Oral Biology 43 (1998) 629±632
composition, or proteolytic degradability of arti®cially
modi®ed teeth, proteins, or amino acids in relation to
the same properties of carious teeth (Armstrong, 1964;
Dreizen et al., 1964). The sugar±protein reaction is
named the Maillard reaction and encompasses a huge
range of intermediates and products. It is also known
as glycation or non-enzymatic browning. The Maillard
reaction in human tissues is associated with the com-
plications of diabetes and ageing, which include vascu-
lar sti€ening, atherosclerosis and renal insuciency.
The reaction has been studied especially in the ®eld of
food chemistry, where it causes characteristic changes
such as the browning of bread during baking. The
brown pigment is formed in a late stage of the reac-
tion. Its exact molecular structure is still unknown.
A di€erent approach to solving the mechanism of
browning was the puri®cation of the brown pigment
formed in carious dentine. It was noted that during
protein hydrolysis in concentrated acid solutions a
dark precipitate formed. This precipitate was thought
to contain the pigment of carious lesions. The elemen-
tal composition of the precipitate resembled that of a
synthetic Maillard pigment (Dreizen and Spies, 1950).
It is uncertain whether the harsh treatment by acid hy-
drolysis could cause arti®cial pigment formation
(Armstrong, 1964). Later, a pigment was isolated from
carious lesions without acid hydrolysis (Engel, 1971).
The infrared spectrum of the isolate resembled that of
a Maillard pigment. It showed one absorption band
that was absent in sound dentine, characteristic of car-
bonyl groups, and marked bands for aromatic double
bonds. Spectroscopy does not seem to be appropriate
to distinguish melanins from Maillard pigments, as
they have some properties in common. Additional indi-
cators of the Maillard reaction would therefore be
needed. Two such indicators have been observed in
carious dentine: a glycosylated peptide (Armstrong,
1968) and hexitollysine (Kuboki et al., 1977). This evi-
dence has been the most speci®c in my view, and indi-
cates that the initial products of the Maillard reaction
are formed in a carious lesion. Further research should
also include indicators for the advanced stage of the
reaction, in which pigment formation takes place.
A number of studies present histochemical evidence
for the presence of melanins in carious lesions. This
evidence is insucient for two reasons. First, di€erent
locations of the melanins are reported: circumventing
the lesion (Opdyke, 1962), di€use throughout the
lesion (Ermin, 1968), and at the lesion surface (Meyer
and Baume, 1966). Second, the silver stain employed
in these studies cannot distinguish melanins from pig-
ments such as lipofuscins and bile acids. Lipofuscins
are formed from the oxidation of lipid molecules.
Their formation from bacterial lipids in carious lesions
cannot be excluded. There is only one report referring
to lipid oxidation in carious material (Dirksen, 1963).
In addition to pigments, reductive substances in the
dentine underlying a carious cavity reduce silver stains
as well, even at an acid pH (Steinman et al., 1959).
Additional stains should be used, such as Nile Blue A,
which stains lipofuscins but not melanins.
The above-mentioned pigments are formed from
chemical reactions within a carious lesion. External
pigments may represent another source of lesion stain.
Kidd et al. (1990) demonstrated that carious lesions do
indeed take up food dyes in vitro. To my knowledge,
no data exist on the presence of food dyes in carious
lesions in vivo. In addition, the lesion may take up
metal ions, which form black precipitates with either
bacterially formed sulphides or protein sulphide
groups. Contamination of mineral by metal ions
during remineralization may be another cause of dis-
coloration. Published results on the presence of metal
ions in carious lesions are contradictory. Di€erent
metals were found in higher amounts in carious lesions
than in sound tissue: iron (Torell, 1957a,b), zinc and
copper (Little and Steadman, 1966), and manganese
(Bao et al., 1990). Malone et al. (1966) observed only
trace amounts of metals in carious and sound dentine.
This indicates that the presence of metal ions is rather
circumstantial, perhaps depending on the diet.
Some bacteria identi®ed in carious material are
known to form pigments. For example, propionic-acid
bacteria (Lee et al., 1978) and black-pigmented
Porphyromonas (Shah et al., 1979) produce haem pig-
ments. An Actinomyces strain isolated from a carious
lesion formed a brown pigment (Hurst et al., 1948).
Boue et al. (1987), however, found no correlation
between lesion discoloration and the presence of
Porphyromonas gingivalis. Neither did Bjùrndal et al.
(1997) ®nd black-pigmented bacteria in every black
lesion.
Based on the results cited above, no ®rm conclusion
is possible on the mechanism essential for carious
tooth discoloration. There is, however, one stage in the
caries process for which a particular mechanism seems
accountable: in dentine caries, the discoloration pre-
cedes the bacteria that penetrate the demineralized
dentine (Fusuyama et al., 1966). It is likely that this
colour change is brought about by compounds di€us-
ing ahead of the bacteria. Because no relation has been
found between lesion pigment and either pigmented
bacteria or metal ions, the remaining possibilities are
the Maillard reaction and the formation of either mela-
nin or lipofuscin. Oxidation is essential for melanin
and lipofuscin formation, which is therefore unlikely in
this anaerobic environment. Although oxidation pro-
motes the transformation of initial Maillard products
into brown polymers, small aldehydes can react with
proteins under anaerobiosis and, unlike carbohydrates
such as glucose, cause browning. In addition, small
aldehydes are also much more reactive than glucose,
 G. A. Kleter / Archives of Oral Biology 43 (1998) 629±632
especially at pH < 7. In the anaerobic and acidic en-
vironment at the lesion front, the Maillard reaction
seems therefore most likely to occur with small alde-
hydes derived from bacterial metabolism. The matrix
would darken while the lesion front progresses with
time. The outer layers, which have been subjected to the
Maillard reaction longer than the lesion front, would
therefore appear darker, in accordance with clinical ob-
servations. It cannot be excluded that with lesion pro-
gression, melanin and lipofuscin will add to the
discoloration because the outer layers have shifted from
anaerobiosis to aerobiosis, allowing oxidation to occur.
Recently, the Maillard reaction in carious dentine
was investigated in more detail. The content of
Maillard products increases as does the Maillard-re-
lated ¯uorescence (lex 370 nm, lem 440 nm). Because
no furosine was found, an established marker of the
initial reaction between glucose and proteins, the reac-
tion probably proceeds through precursors other than
glucose (Kleter et al., 1998). Kuboki's ®nding that hex-
itollysine increases in carious dentine seems contradic-
tory to this result (Kuboki et al., 1977). It can,
however, be accounted for by the reaction between
lysyl aldehyde, which increases in carious dentine
(Kuboki et al., 1977), and glucosamine, instead of
lysine and a hexose. The product of such a reaction
would yield hexitollysine upon reduction, but no furo-
sine after acid protein hydrolysis. In an in vitro model
reaction, demineralized dentine reacted with glucose,
acquiring yellow stain, after which it appeared to be
less sensitive to degradation by pepsin, but not by
trypsin. This indicated a change in collagen structure
caused by glucose (Kleter et al., 1997).
In conclusion, there are clear indications that the
Maillard reaction contributes to lesion discoloration. A
number of Maillard products have been elucidated
recently, which are formed in tissues during ageing and
diabetes. This will enable more detailed future studies
on intermediates and products in carious lesions. In
addition, Maillard-reaction inhibitors are receiving
increased attention in diabetes research. These inhibi-
tors include antioxidants (Ceriello et al., 1992) and
aminoguanidine, which inhibits the transformation of
initial to advanced Maillard products (Brownlee et al.,
1986). Aminoguanidine is currently being tested clini-
cally for the prevention of diabetic nephropathy.
Inhibitors of the Maillard reaction can also help to
prevent the unaesthetic discoloration of root carious
lesions, which are preferably not restored.
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