Cytotoxic Effects of Five Commonly Abused Skin Toning (Bleaching)
Creams on Allium cepa Root Tip Mitosis
O .S. Udengwu
J. C. Chukwujekwu
The Allium test was used to study the cytotoxic
effects of five commonly abused skin toning creams-Ikb, Tura, Top gel,
Dorot and Mililo. These creams are commonly used by some black skinned
people (especially the females) as skin lightening (bleaching) agents.
The results showed that all the five bleaching creams were mito-depressive
in action. They exhibited both chromatoclassic and mitoclassic effects.
Their depressive effects were found to increase with duration of treatment.
The induced abnormalities included chromosome contraction, spindle breakages,
c-metaphase, star anaphase, chromosome stickiness and sticky bridges,
precocious chromosome movement as well as endomitosis. It is suggested
that since all eukaryotic cells are basically the same, these observed
abnormalities could be similar to the effects these chemicals have on
human skin when they are applied. Some of these are known to cause alteration
in melanin formation as well as the biosynthesis of the enzyme tyrosinase.
Furthermore, since certain points on the chromosomes called fragile sites
have been implicated in oncogenesis, the observed abnormalities may be
part of (or include) the switching on mechanisms of such genes, which
could be responsible for the transformation of normal skin cells to malignant
cells in those who abuse these creams.
A practice persists where some dark skinned people, especially the women
folk, abuse skin toning creams in their bid to bleach their skin for better
look and acceptance, despite the fact that many researchers like Barsh
(2003), Fitzpatrick (1988), Lin and Fisher (2007) and Radhakrishnan et
al. (2007) have written about the protective functions of the black
skin against the damaging effects of UV irradiation; which could cause
sunburn and skin cancer for exposed vulnerable skins. The black skin is
able to perform this protective function due to the physical barrier imposed
by the epidermal melanin.
Despite the fact that several researchers, like Bernstein et al.
(1970), Forbes et al. (1970), Elmett et al. (1977),
Parrish et al. (1978), Bergstresser (1989), Cohn and Emmett
(1978), Harber et al. (1982), Bickers (1988) and Krutman and Emmett
(1988) have written about the inherent health hazards which the use of
bleaching creams pose for the users, not much research study has been
carried out to throw adequate light on the nature of the possible cytotoxic
and genetic complications they could cause those who use them.
Available literature indicate that related studies done in this area
had to do with the study of the general effects of some of the chemical
compounds used in the making of some of these bleaching creams on human
skin. Such studies revealed that chemicals like, hydrogen peroxide preparations,
ammoniated mercury, phenols and catechols including monobenzyl ether of
hydroquinone, monomethyl ether of hydroquinone, (p-hydroxyanisole), p-tertiary
butyl phenol, p-tertiary amylphenol and 4 -tertiary butyl catechol could
act as demelanizing agents (Parrish et al., 1978; Douglas, 1980;
Cohn and Emmett, 1978, Marzulli and Maibach, 1980; Anderson and Parrish,
1981; Krutman and Emmett, 1988).
As a result of its critical role in melanin biosynthesis, the enzyme
tyrosinase has become a major target for inhibition in skin-lightening
cosmetics. Hydroquinone is one of the most popular depigmenting agents
and is used extensively to treat several hyper pigmentation disorders.
Depigmentation by hydroquinone is because of its ability to inhibit tyrosinase
as well as its cytotoxicity to melanocytes. However, because of its carcinogenic
properties, use of hydroquinone is banned or limited in cosmetic products
in many countries (Radhakrishnan et al., 2007).
The health and social problems caused by the use of bleaching creams
were compounded following the introduction of topical steroid in 1951
and super potent steroid in 1974 (Frumess and Lewis, 1957; Mihan and Ayres,
1964; Sneddon, 1969; Leyden et al., 1974; Ljubojeviae et al.,
2002; Rathi, 2006).
Apart from the above reported research on the effects of some of the
chemicals on human skin, there are also some cytological studies done
by past researchers that revealed many cytological effects of some other
chemicals on dividing cells of plants. Deysson (1968), Torkowska (1971),
Gulati et al. (1975), El-Bayoumi et al. (1979), Shehab (1979),
Amer and Enaam (1980), Kabirity and Malallah (1980) and Ene-Obong and
Amadi (1987) reported that many glycosides, plant alkaloids, pesticides
and some other chemicals have some cytological effects on root tip mitosis
of experimental plants like Allium cepa, Vicia faba,
Lens esculenta and some other plants. Their effects could generally
be described as mitodepressive, mitopromotive, mitoclassic and chromatoclassic.
The cytological abnormalities they induced include, accumulation of prophase
at the expense of other phases, stickiness of chromosomes, contraction
of chromosomes and sticky bridges, lagging of chromosomes (Raj and Shubba,
1971; Torkowska, 1971; Shehab, 1979; Kabarity and Malallah, 1980; Ene-Obong
and Amadi, 1987). Other effects include spindle disturbances, polyploidy,
chromosome breakages, pycnosis, binucleate cells, chromosome denaturation
(Raj and Shubba, 1971; Torkowska, 1971; Shantharmurthy and Rangaswamy,
1979; Shehab, 1979; Ene-Obong and Amadi, 1987).
Similar studies with some extracted known alkaloids like Colchicine,
Podophy-lotoxin, Coumarin, Vinblastine, Vincristine and Mimosine exhibited
remarkable cytological effects which include sticky bridges by colchicine
spindle breakage by Vinblastine spindle damage, stickiness and sticky
bridges, laggards, precocious chromosomes by podophyllin and mimosine
(Deysson, 1968); spindle formation inhibition by colchicines (Inone ,
The aim of this present study was to ascertain the nature of the cytotoxic
effects of these bleaching creams on the building blocks of life- the
cells, which contain the chromosomes and which in turn contain the genes
that control all the biological activities of an organism. It is believed
that since the structure of the cells of all eukaryotic cells are basically
the same, coupled with the fact that it is easier to study with plant
materials, the observed cytological effects of these bleaching creams
studied, with the Allium test, may throw some light on the possible
mode of action of the chemical constituents of these creams on the cells
of the human skin in changing melanin present in melanosomes from the
dark- coloured oxidized form to the lighter coloured reduced form, interference
with the biosynthesis of melanin, prevention of the biosynthesis of tyrosinase,
premature skin aging, dermatitis and carcinogenesis as noted by Douglas
(1980), Lin and Fisher (2007) and Radhakrishnan et al. (2007).
The Allium test is acclaimed to be a very cheap and sensitive tool
for the detection of potentially genotoxic substances (Fiskesjo, 1985;
Sabti, 1989; Smaka-Kinkl et al., 1996; Chang et al., 1997;
Rank and Nielson, 1998; Cotelle et al., 1999; Moraes and Jordao,
2001). The findings may also help to caution the users of such creams
about the potential dangers they might be exposing themselves to.
MATERIALS AND METHODS
This research was initiated in 1994 and concluded in 2007 in the Department
of Botany, University of Nigeria, Nsukka. Rooted onions Allium cepa
(2 n = 16) bulbs were used to investigate the cytotoxic effects of Top
gel, Ikb, Tura, Dorot and Mililo which are among the commonly abused commercial
bleaching creams. Table 1 gives the chemical composition of the creams
as indicated on the cream containers by the manufacturers. One hundred
and eight medium sized, fresh red onion bulbs, each weighing approximately
85 g, bought from the Nsukka market, were grown in water soaked, well
cured, Gmelina wood saw dust, in wooden germination boxes in the
Botanical garden, University of Nigeria, Nsukka. When the roots were about
2-5 cm long, after about 4-6 days of planting, the rooted bulbs were transferred
to 100 mL beakers containing distilled water and left for 24 h in order
to allow enough time for recovery, in case there were any abnormalities
caused by the sawdust culture.
||Chemical composition of the five bleaching creams
Treatment procedure and duration of treatment:
||Time range and duration of treatments (h)
The one hundred
and eight bulbs were divided into 6 groups, with three bulbs set for each
of the six treatment durations for each of the creams and the water control.
The onion roots were evenly rubbed with each of the bleaching creams, for
the different treatments, just like humans rub creams on their skin. The
roots were covered with moistened cotton wool to enhance absorption of the
creams by the roots, before suspending the bulbs in
150 mL beakers.
Three bulbs, for each treatment (duration), were retained in distilled water,
in a 150 mL beaker, without any cream, to serve as the control. Table 2
gives a summary of the durations and times of treatment.
Fixation and hydrolysis of the root tips: At the end of each treatment
period, four healthy roots were cut off from each of the three bulbs for
each of the treatment durations and for each of the creams as well as
the water control. The roots were washed in distilled water for 2-3 times
and then fixed in Carnoy`s solution (1:3 acetic acid:absolute alcohol).
The fixed materials were kept in the refrigerator for at least
24 h after which they were stored in 70% alcohol before usage. The root
tips were hydrolysed in I N HCl at an acid temperature of 60oC
for 5-7 min using a Gallenkamp water bath.
Slide preparation and study: Hydrolysed root tips were then washed
2-3 times in tap water, sliced and squashed on a clean glass slide in
Lacto Proprionic Orcein (LPO) and left for 5 min so, that the stain will
be absorbed by the chromosomes. The temporary slides were then studied
under the microscope. Good preparations were sealed off using nail varnish.
Good plates were photographed with Leitz Ortholux II microscope at 1000
magnification and the prints were done at 4x negative enlargements.
For making the cell counts, three slides were prepared for each treatment
duration and for each cream and the water control. Different fields were
picked at random with the three different slides and the views of interests
scored. The number of cells counted ranged between 3,000 and 3,200.
Data collections and analysis: After observing, counting and recording
dividing cells and total number of cells from 12 different fields for
each cream for each treatment duration, the mitotic indexes were calculated
using the formula below. Thereafter the means and standard errors of the
mitotic indexes for each of the creams as well as the treatment durations
Other computations that were done based on classification of cytological
For all analysis of variance the Randomized Complete Block Design (RCBD)
was used. Three main effects and three first order interactions were analyzed
for the analysis of variance of the dividing cells.
RESULTS AND DISCUSSION
The Allium cepa root tips treated with the five bleaching creams
(Ikb, Tura, Dorot, Top-gel and Mililo) exhibited many types of abnormalities.
These abnormalities involved all stages of mitosis. Generally, all the
five bleaching creams induced mitodepressive effects i.e., the reduction
in number of dividing cells. The bleaching creams showed different degrees
of depression based on duration of treatment. On the average, the highest
degree of depression was scored by Tura followed by Dorot, Top-gel, Mililo
and the least by IKB. It was found that increase in duration of
treatment affected this depression. For all the bleaching creams the trend
of depression showed highest reduction of mitotic indexes at the longest
durations of treatment of 24 h (Table 3). The reason for Tura being the
most mitodepressive may not be unconnected with the fact that essentially
it contains hydroquinione mixed with allantoin. The effects of hydroquinone
on human skin have engaged the attention of dermatologists for a long
time. For Ikb, which showed the least mitodepression, the presence of
vitamin E as one of its constituents, may have mitigated the effect of
hydroquinone and allantoin on the root tip cells. Vitamin E is known to
play vital roles in the formation of red blood cells, muscles and other
tissues and in preventing the oxidation of vitamin A and fats. It is also
popularly advocated for a wide range of diseases.
||Effect of treatment duration on the mitotic index of Allium cepa
root tips treated with five bleaching creams
||Percentage mean No. of dividing cells at different phases of mitosis
|Means followed by the same letter(s) in each column
or row are not significantly different at 5% level using LSD
||Analysis of variance of dividing cells
|**: Significant at p<0.01, ***: Significant at p<0.001
The effect on the mitotic index also affected the mean percentage number
of cells at different phases. Significant increase in percentage of prophase
phase with significant corresponding decrease in other phases was observed
This was found to be highest with Top-gel followed by Tura, Ikb, Mililo
and least with Dorot. A possible reason for the accumulation of prophase
at the expense of the other phases could be due to the ability of the
constituents of these bleaching creams to attack and disrupt the spindle
apparatus that are normally formed prior to the cell transiting into metaphase.
Analysis of variance of the dividing cells as given in Table 5 indicate
that there was a very highly significant difference between the treatments
The differences between the mitotic stages on one hand and the durations
of treatment on the other were very highly significant (p<0.001). The
first order interaction on Table 5 (i.e., Treatment x Mitotic stage showed
a very highly significant difference (p<0.001), the last interaction
(i.e., Mitotic stages x Duration) also showed a highly significance difference
(p<0.01). In the case of mitodepression, the degree of depression varied
with the creams and this can be attributed to their chemical components
as shown in Table 1. Similar depressions were also observed by some researchers
using chemicals like 2,4,- D, Amitrols, Phenols, Isoprophyl and some plant
alkaloids like colchicine, podophylotoxin, Coumarin, Vinblastine etc.
(Amer and Farah, 1974; Amer and Enaam, 1980; Kabarity and Malallah, 1980;
Ene-Obong and Amadi, 1987). They attributed the phenomenon to the inhibition
of DNA replication at interphase during the S-phase leading to inhibition
of other mitotic stages.
It is worth noting that the observed mitodepressive actions of these
creams can be largely attributed to hyroquinone, which is the major chemical
component of almost all the creams (Table 1). It has been reported that
hydroquinone inhibits the synthesis of melanin that protects the skin
from UV radiation in a process known as de-pigmentation (Douglas, 1980;
Lin and Fisher, 2007; Radhakrishnan et al., 2007). Its mitodepressive
ability, as the major component of the five creams suggests that one of
the possible processes through which it causes de-pigmentation in human
skin could be through the suppression of the mitotic process needed for
the replacement of worn out melanocytes.
||Percentage mean No. of dividing cells showing abnormalities after
treatment with five bleaching creams for different time durations
|Means followed by the same superscript letters in each
column or row are not significantly different at 5% level using LSD
||Percentage mean No. of abnormal cells at different phases of mitosis
Means followed by the same superscript letter(s)
in each column or row are not significantly different at 5% level
With serious reduction in the amount of new melanocytes being formed
(mitodepression), the skin may gradually loose its characteristic black
colour resulting to a fair skin appearance for the user with the attendant
exposure of the skin to ultraviolet radiations and other potential infections.
Table 3 shows that mitodepression increased with duration of treatment
and this may account for the fact that the longer people used these creams,
the lighter and more tender their skin become and in cases of excessive
use the skin may loose its ability to perform its primary protective functions.
Additionally, it has been observed that once users stop the application
of these creams, nature fights back to restore the status quo by stimulating
rapid cell division among the melanocytes. Unfortunately such natural
response does not restore the status quo, but rather the skin of the individual
becomes darker and less attractive, hence the tendency to continue usage
or even look for stronger brands which may eventually lead to malignancy
of the melanocytes.
Table 6 indicates that there were no significant differences between
the induced abnormalities by the five creams over the different time durations.
A possible reason for this could be that the creams being powerful de-pigmenting
agents were able to cause different damages to the genetic materials in
the cells shortly after their application. These quick damages however
appear not be influenced by duration of treatment, unlike the situation
with mitodepression. This observation may account for the frequent application
of these creams by their users as well as the observable lightening of
the skin a few days after commencement of their application.
Table 7 shows that most of the abnormalities were scored at the
prophase and metaphase stages and least at anaphase and telophase stages.
The different types of abnormalities observed were mostly of the sticky
(chromotoclassic) and disturbed (mitoclassic types). Figure
1a-f shows that some of the abnormalities observed included disturbed
prophase, sticky metaphase and anaphase, c-metaphase, star anaphase and
anaphase with precocious chromosomes. Table 8 indicates
that disturbed phases, stickiness, anaphase bridges and to a less extent
precocious chromosome movements at anaphase and star anaphase were the
most frequent abnormalities encountered. The stickiness types occurred
in the form of sticky metaphase, anaphase and telophase. It was also found
that the degree of stickiness varied with the treatments (creams) for
example Ikb gave the highest degree of stickiness followed by Top-gel,
Tura and the least by Dorot and Mililo. This type of anomaly has been
interpreted to be due to the depolymerization of DNA, partial dissolution
of nucleoproteins, breakage and exchange of the basic folder fibre unit
of chromatids and the stripping of the protein covering of DNA in chromosomes
(Mercykutty and Stephen, 1980). This anomaly was also reported to be induced
by phosphonothioate insecticide, leptophos, on Vicia faba (Ali
and Amer, 1974; Amer and Farah, 1974; Shehab, 1979).
||(a) Disturbed prophase (b) Mildly sticky metaphase (left
arrow) and Early anaphase (right arrow) (c) C-metaphase (d) Very sticky
anaphase (arrow) (e) Star anaphase and (f) Anaphase with precocious
||Percentages of the main types of abnormalities observed at different
|Distur: Disturbed, Endo: Endomitosis, Preco: Precocious
The disturbed types of abnormalities included; disturbed prophase, metaphase,
anaphase and telophase. The degree of disturbance varied with the creams,
for example Mililo gave the highest degree of mitoclassic effect followed
by Ikb, Tura, Dorot and the least was scored with Top-gel. The disturbed
prophase observed by earlier researchers was attributed to irregular arrangement
of chromatin threads (Shehab, 1979). However disturbed metaphase and the
later phases are believed to be as a result of spindle formation inhibition
or damage (Borisy and Taylor, 1967a, b; Deysson, 1968; Pritchard and Court,
1968). This spindle disturbance during the metaphase stage resulted to
the scattering of the chromosomes within the cell, thus preventing the
chromosomes from moving towards the opposite poles during anaphase leading
to what is referred to as c-metaphase (Fig. 1c) (Weisenberg
et al., 1967; Torkowska, 1971; Artvinli, 1987).
The consequences of these observed abnormalities are often interrelated.
According to Fiskesjo (1985), c-mitosis is regarded as indicative of a
weak toxic effect which may be reversible, a vagrant chromosome, a weak
c-mitotic effect indicating risk of aneuploidy, while sticky chromosomes
indicate a highly toxic, irreversible effect, probably leading to cell
death. Anueploidy has been reported to probably be the only mutation that
can explain all aspects of carcinogenesis (Duesberg and Rasnick, 2000).
Endomitosis or endopolyploidy was another abnormality observed. It was
observed only in metaphase stage. It occurred in almost all the treatments
with highest frequency occurrence in Dorot followed by Top-gel, Ikb, Mililo
and least by Tura. This abnormality could have arisen as a result of inhibition
of spindle mechanism leading to the arrested phase reverting to the interphase
stage (Nelson, 1972; Ene-Obong and Amadi, 1987). Single and multiple bridges
were observed in nearly all the treatments. They occurred only in anaphase
and telophase stages. The degree of this anomaly also varied with different
treatments (creams). Top-gel gave the highest degree of bridges followed
by Ikb, Dorot, Tura and the least by Mililo. The incidence of bridges
have been attributed to the sticky nature of chromosomes that brought
about non-synchronization at separation of chromatids during the movement
of chromosomes towards the poles, such chromosomal bridges were also reported
by Amer and Farah (1974) and Kabarity and Malallah (1980).
Other rare abnormalities observed include precocious chromosomes movements
at anaphase, which is believed to be caused by the non-synchronization
of the spindles in their poleward movement during anaphase or due to early
disjunction of a pair of chromatids such that one starts off its poleward
journey earlier than others (Shehab, 1979; Sarbhoy, 1980). Star anaphase
(Fig. 1e), could be attributed to disorientation of
the chromosome spindles with the result that the centromeres all point
towards a centre in a circular form instead of in the direction of the
poles. Few cases of chromosomal breakages were also observed with Tura
which could be linked to the actions of hydroquinone, the major component
of the cream, on the chromosomes. Hydroquinone according to Radhakrishhnan
et al. (2007) has carcinogenic properties and hence its use is
banned or limited in cosmetic products in many countries. Breakages on
certain locations on the chromosomes called fragile sites that contain
oncogenes have been linked with cancer (Yunis and Soreng, 1984; De Braekeleer
et al., 1985; LeBeau, 1986; Pellicia and Rocchi, 1986; Yunis et
Incidences of skin cancer in those who use these creams may therefore
not be unconnected with the actions of hydroquinone on fragile sites of
It has been shown that these creams apart from being mitodepressive also
exhibit both mitoclassic and chromatoclassic effects. These effects were
found to depend on individual creams and duration of treatment. This duration
dependent mitodepressive actions could be attributed to the different
chemical compositions (i.e., hydroquinone, fluocinonide, cetrimide, allantoin,
propylene glycol, irgassan etc) of the creams. It is suspected that these
chromatoclassic and mitoclassic effects could be close to their mode of
action in the alteration of melanin formation and inactivation or the
prevention of the biosynthesis of the enzyme tyrosinase in humans.
It is postulated that since all eukaryotic cells are basically the same,
these observed anomalies with plant cells are expected to be similar,
if not more pronounced, with the animal (human) cells which lack rigid
cell walls. Studies by different researchers indicate that the Allium
test is a very sensitive tool for the detection of potentially
genotoxic substances (Fiskesjo, 1985; Sabti, 1989; Smaka-Kinkl et al.,
1996; Chang et al., 1997; Rank and Nielson, 1998; Cotelle et
al., 1999; Moraes and Jordao, 2001). The protocol of rubbing the creams
on the roots the way humans rub these creams on their bodies, as well
as the observed cytotoxic effects even after short durations of application,
suggests that the Allium test could be a pertinent tool for a better
understanding of the cytotoxic problems and complications the abuse of
these creams could cause.
Finally, since this study has shown that the degree of mitodepression
increased with duration of treatment as well as with the nature of the
chemical constituents of these creams; this could be related to the observed
fact that the skins of the abusers of these creams show severe deterioration
with prolonged usage. Incidentally, to shorten the action time of these
creams, some manufacturers now produce more powerful creams whose bleaching
effects, together with its concomitant degradation of the skin, manifests
after shorter periods of time. From the cytological point of view, such
a development may further complicate the social and medical problems the
abuses of these products are likely to cause.
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