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Journal of Applied Sciences and Environmental Management
World Bank assisted National Agricultural Research Project (NARP) - University of Port Harcourt
ISSN: 1119-8362
Vol. 10, Num. 3, 2006, pp. 159-162
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Journal of Applied Sciences & Environmental Management, Vol. 10, No. 3, September, 2006, pp. 163-166
The Effect of Two African
Mineral Dyes on the Activity of Alkaline Phosphatase in the Skin and Serum of
Albino Rats
1ADEBAYO,
GB, 2SUNMONU,T O, 3ADEKOLA, FA; 3OLATUNJI ,GA
1Department of Chemistry, KwaraState Polytechnic,
Ilorin, P. M. B 1375, , Ilorin, Nigeria.
2Department
of Biochemistry, University of Ilorin, P. M. B. 1515, , Ilorin, Nigeria.
3Department
of Chemistry, university of Ilorin, P. M. B. 1515, , IlorinNigeria
Code Number: ja06071
ABSTRACT: The
effect of two African mineral dyes Yombo tumtum (YT) and Yombo fita (YF) on the
activity of alkaline phosphatase (ALP) in the skin and serum of albino rats was
investigated. The chemical analyses of the dyes were first carried out using
solubility test, pH determination and X-ray fluorescence (XRF) elemental analysis.
Six different concentrations (0.05, 0.15, 0.25, 0.5, 0.75 and 1.00%) of each
dye were prepared using hydrogen peroxide as solvent. A total of 70 albino rats
(Rattus norvegicus) were used for the study. The rats were grouped into
14 of 5 rats and were maintained on commercial feed for a period of 30 days,
during which the experiment lasted. Group I served as the first control
and the animals had distilled water applied on their heads. Group 2 which
served as the second control contained rats on whose heads were applied the
dissolving solvent i.e. hydrogen peroxide. Rats in group 3 to 8 had the various
concentrations of YT (black) dye applied on their heads, while those in groups
9 to 14 had the various prepared concentrations of YF (white) dye applied on
their heads. At the end of the experiment, blood samples were collected and
portion of the head skin where the dyes were applied was cut. The activity of
ALP was thereafter determined in the serum and skin of the experimental
animals. The results revealed a significant decrease (p<0.05) in the
activity of ALP as the concentration of the dye increased in both the serum and
skin @JASEM.
The
use of hair dye is fast gaining popularity especially among ladies and old men.
Young ladies engage in the use of dyes mainly for fashion while old men use
dyes to mask their grey hair. Dyes are basically of two types namely natural
dyes and synthetic dyes. Natural dyes include plant dyes, animals and mineral
dyes. Plant dyes can be obtained from roots, flowers, leaves, fruits and bark
of plants such as wood and indigo. Some animal sources of dyes include insects
such as Cochineal, found on Cacti in Mexico, Lac
found in India and Iran and
kermes, found on oak tree near the Mediterranean.
Mineral dyes come from ocher (yellow, brown, red); limestone or lime (white),
manganese (black), cinnabar and lead oxide (red) azurite and lapis lazuli
(blue), and Malachite (green)( Wayne, 1994). The use of synthetic
dyes became paramount in the mid-nineteenth century when the demand for
hand-made rugs increased in the West. The need for easy to use and less
expensive dyes with wider range of colours led to the development of synthetic
dyes in Europe especially in Germany. The
first synthetic dye, Fuchsine (magenta aniline) was developed in the 1850s.
Other synthetic aniline dyes such as chrome dyes were later developed.
Hair
dyes can be either temporary or permanent dyes with varying chemicals which may
be poisonous to the users. Temporary dyes contain silver, mercury, lead, arsenic, bismuth,
paragallol and denatured alcohol while permanent dyes contain naphthylamine,
phenylenediamines, toluene-diamines and other aromatic compounds. Most people
dye their hair without considering the side effects of the chemical
constituents of the dye. The decision to change hair colour has recently become
more complicated due to some recent studies which linked hair colouring with an
increase risk of contacting certain cancers (Patlak, 1993).
Several other studies on dyes have been carried out by various researchers
which implicated dyes as carcinogens (Coldiz, 1994, Chase, 1998, Oncolink,
1999). It has also been reported that hair dyes may be hazardous to childrens
health due to the presence of lead in the form of lead acetate, as exposure to
such lead-containing hair dyes can cause neurological problems (Marcus, 1997).
Other symptoms such as local skin irritation can be associated with toxic
substances in the hair dyes (Coldiz , 1994). Alkaline phosphatase
is an enzyme, or more precisely a family of related enzymes, produced in the
bile ducts, intestine, kidney, placenta and bone. It is mostly found in an
alkaline state with a pH of 9. It is used extensively as a tumor maker and also
present in bone injury, pregnancy, or skeletal growth. An elevation in the
level of serum alkaline phosphatase suggests disease of the bile ducts. Serum
alkaline phosphatase activity can be markedly elevated in bile duct obstruction
or in bile duct diseases such as primary biliary, cirrhosis or primary
sclerosing cholangitis. ALP is also a marker enzyme for the plasma membrane
and endoplasmic reticulum (Akanji, et al, 1993). Therefore damage to the
membrane especially in the skin may adversely affect the activity of ALP.
The
present study is therefore aimed at exploiting the activity of alkaline
phosphatase (ALP) which is a membrane-bound enzyme as a possible means of
understanding the toxicological effect and the mechanism of action of two
African mineral dyes YT (black) and YF (white) which are commonly used in Nigeria for
dyeing hair.
MATERIALS AND METHODS
Source
of the dye:
Two samples of rock-like mineral dyes were obtained from
Emirs Market in Ilorin, Nigeria. The
Local names of the dyes are Yombo-Tumtum (YT) for black and Yombo-fita (YF) for
white. The original source of the samples was traced to the Southern part of Ghana.
Solubility
test:The solubility of each dye sample was investigated in
fourteen different solvents. The solvents include deionized water, hydrogen
perioxide, Acetone, benzaldehyde, acetaldehyde, ethanol, petroleum ether,
concentrated HNO3, concentrated HCl, methanol, diethyl ether, ethyl
acetate, acetic acid and aniline. The test was carried out by adding 10mg of
the well ground (<80µm) to 5cm3 of each of the above solvents in
a test tube at room temperatures. Where the dye was not soluble in the cold, a
gentle heat was applied placing the test tube inside a heated water-bath
(Adetuyi et. al., 2003).
Elemental
analysis of the dye: Each sample was crushed and ground in a Tungsten Carbide Spex
Mill followed by other procedures specific for the method of analysis (Potts,
1993). Each sample was then analyzed for trace elements using Energy Dispersive
Xray Fluorescence (ED-XRF) Spectrophotometer. The EDXRF facility consists of
two interchangeable (55Fe and 109Cd). Annular source, a Canberra
model SL 12170 silicon solid state detector and the associated pulse processing
electronic which are coupled to ADC-Card. The facility runs on PC with Maestro
software for spectra acquisition. Sensitivity calibration was done using thick
pure metal foils (Ti, Fe, Co, Ni, Cu, Zn, Zr, Nb, Mo, Sn, Ta, Pb) and stable
analytical grade chemical compound K2CO3, CaCO3,
Ce2O3, WO3, ThO2 and U3O8.
Quantification of the concentrations of detectable elements was done using a
modified version of emission transmission method (Potts et. al.,
1984).
Preparation
of different concentrations of the dyes: Six different
concentrations of each dye (YT and YF) were prepared (0.05, 0.15, 0.25, 0.50,
0.75 and 1.00%w/v) using Hydrogen peroxide as the dissolving solvent. For
example, 0.05% concentration was prepared by dissolving 0.05g of the dye in
100cm3 of hydrogen peroxide in 100cm3 volumetric flask.
Experimental
Animals:A
total of 70 albino rats (Rattus norvegicus) with an average
weight of 26.8g were used for the study. The rats were grouped into 14 of 5
rats each as follows.
Group
1: Rats on whose heads were applied 2cm3 distilled water (Control 1)
Group
2: Rats on whose head were applied 2cm3 of dissolving solvent (H202)
Group
3: Rats on whose heads were applied 2cm3 of 0.05% conc. YT dye
Group
4: Rats on whose heads were applied 2cm3 of 0.15% conc. YT dye
Group
5: Rats on whose heads were applied 2cm3 of 0.25% conc. YT dye
Group
6: Rats on whose heads were applied 2cm3 of 0.50% conc. YT dye
Group
7: Rats on whose heads were applied 2cm3 of 0.75% conc. YT dye
Group
8: Rats on whose heads were applied 2cm3 of 1.00% conc. YT dye
Group
9: Rats on whose heads were applied 2cm3 of 0.05% conc. YF dye
Group
10: Rats on whose heads were applied 2cm3 of 0.15% conc. YF dye
Group
11: Rats on whose heads were applied 2cm3 of 0.25% conc. YF dye
Group
12: Rats on whose heads were applied 2cm3 of 0.50% conc. YF dye
Group
13: Rats on whose heads were applied 2cm3 of 0.75% conc. YF dye
Group
14: Rats on whose heads were applied 2cm3 of 1.00% conc. YF dye
The
different concentrations were applied every three days and the experiment
lasted for 30days. The dye was applied every three days to depict continuous
and prolong usage of the dye. All the rats were housed in separate metabolic
cages and fed ad libitum with commercial rat chow and water. At the
expiration of 30days blood samples were collected and portion of the head skin
where the dyes were applied was cut.
Assay
for Alkaline Phosphatase Activity:The activity of ALP was
assayed in the skin and serum of the rats using the modified method of WrightandPlummer(1974). Two test tubes labelled blank and test were placed in the test tube
rack. 2.2ml of 0.1M carbonate buffer was pipetted into the test tubes. 0.1ml of
0.1M MgSO4.7H2O was added into the test tube.0.2ml
distilled water and homogenate was added into the blank and test tubes
respectively. The mixture was shaken and incubated in water bath at 370C
for 10 minutes. 0.5ml, 19Mm PNPP was added into each of the test tubes and
incubated in water bath at 370C for another 10 minutes. Finally,
2.0ml of 1N NaOH was added into the test tubes and absorbance of test against
blank was taken at 0, 1, 2 and 3 minutes at 400nm. The absorbance readings obtained
were then used to calculate the specific activity of Alkaline
Phosphatase (ALP).
RESULTS AND DISCUSSION
Solubility
Test:The results of the solubility test of the two samples of
dye show that the two dye samples were soluble in almost all the solvents
employed (except petroleum ether and concentrated HCl) with the solution having
different colours. Almost all colours appear to be unstable as they changed
after 24 hours. The samples were found to be soluble in both polar and
non-polar solvents. The solubility of samples may be attributed to the
presence of both organic and inorganic components in the samples. The colour
observed may be attributed to the existence of certain complexes involving some
elements within the sample and organic species acting as ligands. The pH of the
resulting aqueous solution shows that the samples were slightly alkaline. This
may be due to the presence of carbonates or hydrogen carbonate of alkaline and
alkaline earth metals in the samples. The solubility of the samples in both
polar and non-polar solvents probably explains penetrating ability of the dyes
into different surfaces.
Elemental
Xray Flourescence (XRF) Analysis: The
results of XRF analysis of the two samples YT and YF are summarized in Table 1.
Twenty elements were recorded and concentrations range from major to
ultra-trace levels. The major elements include Potassium (K) and Sodium (Na).
Minor elements are Ti, V, Cr, Zn, Mn, and Fe, trace elements Co, Ni, Cu, As, Pb
and Zr and the ultra trace elements include Rb, Sr, Y and Mo. The
results of XRF reveal that these dyes may have certain degree of deleterious
effect due to the presence of toxic metals such as As, Pb, Cr, Co, and Zn-in
the two samples. Some of the elements detected in the dyes are trace elements
which have been found to be potent carcinogens. Studies have shown that
elements such as Cr, Pb, As, and Cu are potent carcinogens (De Zwart and
Sluoff, 1987). This probably account for the implication of heavy metals on the
etiology of cancer.
Specific
Activity of Alkaline Phosphatase (ALP): The
specific activity of ALP in the skin and serum of the rats following the
application of the two samples of the dye is presented in Table 2. The
significant reduction (p<0.05) in the specific activity of the enzyme as the
concentration of the dye increases in the skin and serum may in part be due to
damage to the tissues. ALP is a marker enzyme for the plasma membrane and
endoplasmic reticulum. Therefore damage to the membrane especially in the skin
may adversely affect the activity of ALP. The reduction in ALP activity could
also be attributed to inhibition of the enzyme by the dye as is the case with
consumption of metabisulphite (Akanji et. al., 1993),or
inactivation of the enzyme molecules in situ (Umezawa and Hooper,
1982). The gradual reduction in enzyme activity as the concentration of the dye
increases has clearly demonstrated that the dye has a pronounced effect on the
skin and serum.
Table 1: Elemental
analysis of YT and YF by XRF techniques
S/NO
|
ELEMENT
|
SAMPLE
YT
|
SAMPLE
YF
|
1
|
K
|
0.43(p)
|
0.43(p)
|
2
|
Ca
|
0.26(p)
|
0.26(p)
|
3
|
Ti
|
0.07(p)
|
0.07(p)
|
4
|
V
|
348
|
345
|
5
|
Cr
|
204
|
207
|
6
|
Mn
|
151
|
175
|
7
|
Fe
|
125
|
125
|
8
|
Co
|
66.0
|
66.0
|
9
|
Ni
|
42.0
|
53.0
|
10
|
Cu
|
43.0
|
35.0
|
11
|
Zn
|
123.0
|
26.0
|
12
|
As
|
23.0
|
21.0
|
13
|
Pb
|
33.0
|
31.0
|
14
|
Br
|
8.00
|
13.0
|
15
|
Rb
|
7.00
|
9.00
|
16
|
Sr
|
7.00
|
9.00
|
17
|
Y
|
7.00
|
9.00
|
18
|
Zr
|
11.0
|
6.00
|
19
|
Nb
|
15.0
|
30.0
|
20
|
Mo
|
5.00
|
5.00
|
Note: (p) → Values in
Percentage, others are in ppm.
Table 2: Specific activity of alkaline
phosphatase in the skin and serum of rats following the application of dye
Concentration
of Dye
|
Specific activity mmol/mg protein/min
|
Skin
|
Serum
|
Yombo
Tumtum (YT)
|
Yombo
Fita (YF)
|
Yombo
Tumtum (YT)
|
Yombo
Fita (YF).
|
Control
|
8.74
± 0.09a
|
8.14
± 0.08a
|
8.29
± 0.24a
|
8.02
± 0.02a
|
Solvent
|
6.90
± 0.10 b
|
6.85
± 0.09 b
|
5.46
± 0.00 b
|
5.15
± 0.01 b
|
0.05%
|
5.74
± 0.05c
|
5.21
± 0.05 c
|
4.24
± 0.08 c
|
4.04
± 0.03 c
|
0.15%
|
4.12
± 0.02 d
|
3.91
± 0.02 d
|
3.36
± 0.07 d
|
3.01
± 0.00 d
|
0.25%
|
2.48
± 0.04e
|
2.16
± 0.03e
|
2.72
± 0.09 e
|
2.25
± 0.03 e
|
0.50%
|
1.80
± 0.00f
|
1.30
± 0.00 f
|
2.11
± 0.02 f
|
1.98
± 0.01f
|
0.75%
|
0.99
± 0.03 g
|
0.81
± 0.01g
|
1.54
± 0.01g
|
1.34
± 0.03g
|
1.00%
|
0.42
± 0.02h
|
0.31
± 0.02 h
|
0.97
± 0.05 h
|
0.63
± 0.01 h
|
Note: Each value is a
mean of five determinations ± SEM; a, b, c,
.column values with different
superscripts are significantly different (p< 0.05).
Conclusion: The
dyes have been found to reduce the activity of ALP in the skin and serum of the
rats. Further studies are still in progress especially as relates to the effect
of the dye on the activity of Acid phosphatase which has been implicated in
prostate cancer. The decrease in the activity of ALP may not be unrelated to
the presence of some highly toxic metals such as As, Pb, Cr, Co and Zn in the
studied mineral dye. Further work is presently going on in our laboratory with
respect to isolation, purification and characterization of organic compounds in
the mineral dye.
REFERENCES
- Adetuji, A. O., Popoola, A. V. and Lajide. L. (2003). Isolation and A
colouring potentials of leaf extract of Teak plant (Tectone Grandis). J. Chem.
Soc. Nigeria 28 (1), 34-39.
- Akanji, M.A., Olagoke, O.A. and Oloyede, O. B. (1993).
Effect of chronic consumption of metabisulphite on the integrity of rat liver
cellular system. Toxicol. 81, 173-179.
- Chase, M. (1998). Study shows hair dyes pose scant cancer
risk, Wall Street JournalU.S.A.
- Coldiz, G.A. (1994): Hair dyes and cancer risk J. Natl.
Cancer Inst. 86, 164-165.
- De Zwart, D. and Sluoff, W. (1987). Toxicity of Mixtures of
heavy metals and petrochemicals to Xenopus laevis. Bull. Environ.
Contam. Toxicol. 38, 345-351.
- Marcus, M. B. (1997). Lead in hair dyes. US News Online. US
News World Report Inc.
- Oncolink Team (1999). study sees no link between hair dyes
and lymphomas. The Trustees of the University of Pennsylvania, U.
S. A.
- Patlak, M. (1993). Hair dye dilemmas, US Food and Drug
Administration, U.S.A.
- Potts, P. J. (1993). Energy Dispersive X-ray spectrometry.
A handbook of Silicates Rock Analysis, Blackie, Glasgow, Pp
286-325
- Potts, P.J, Webb, P.C. and Waston, J.S. (1984). Energy
Dispersive X-Ray Fluorescence Analysis of Silicate Rock for Major and Trace
Element. X-Ray Spectrum 13, 2-15.
- Umezewa, H. and Hooper, I. R.
(1982). Aminoglycoside antibiotics. Spanger Verky, Berlin,
Hodelberg New York.
- Wayne, P. A. (1994). Natural dyes. Journal of the American
Botanical Council 32, 30.
- Wright,
P. J. and Plummer, D. T. (1974). The use of urinary enzymes measurement to
detect renal damage caused by nephrotoxic compounds. Biochem. Phermacol. 23,
65-73
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