Thirty seven percent formaldehyde solution, known as formalin, is characterized
as an inexpensive and effective preservative that rapidly penetrates the
tissue. It is frequently used as one of the most common preservatives
for fish. Besides, it is widely used as a disinfectant in many human medicines
and cosmetics and as an antiseptic in veterinary drugs and biologicals
and in fungicides, textiles and embalming fluids (Feick et al.,
2006; Ross et al., 2002; IARC, 1982). In the urban area of Bangladesh,
the most commonly consumed fish is rui (Labeo rohita), majority
of which come from neighboring countries like India and Myanmar. These
rui fish are usually treated with excess formalin prior to import. But
the levels of formalin used in these fish and their possible harmful effects
on body tissues are not known clearly. Some reports suggest that ingested
formaldehyde causes inflammation of the linings of the mouth, throat and
gastrointestinal tract and eventual ulceration and necrosis of the mucous
lining of the gastrointestinal tract (Yanagawa et al., 2007; Sidhu
and Sidhu, 1999; Owen et al., 1990) and in case of chronic exposure,
formaldehyde has the potential to cause cancer and a variety of unknown
pathology (Hildesheim et al., 2001; Vaughan et al., 2000;
Wippermann et al., 1999). Thus, if the fish with high formaldehyde
levels are consumed by human for a long period, they may encounter a host
of biochemical, as well as, pathophysiological abnormalities, although,
to what extent it may cause such abnormalities and subsequent health hazards
have remained unclear.
The objectives of this study were to estimate the levels of formaldehyde
in the rui fish (Labeo rohita) in Bangladesh and to examine the
possible detrimental effects of formaldehyde in rat liver and intestinal
tissues. Toxic compounds may cause cellular damage, which is often exhibited
by the electron rich reactive species, called lipid peroxides (LPO) (Halliwell
et al., 1992). Therefore, by measuring the levels of LPO, the extent
of tissue damage caused by formaldehyde could simply be evaluated.
MATERIALS AND METHODS
Fish: The study was conducted from August, 2005 to February, 2006.
Thirty-five imported rui fish (Labeo rohita) weighing ~2.5 kg were
purchased from 7 different markets of Dhaka city. A similar number of
fresh rui fish of similar size and weight were collected from 7 different
ponds away from Dhaka city. Both the imported and the fresh rui fish were
brought to the laboratory and stored on ice at temperature around 0°C.
Preparation of fish tissue homogenates and estimation of formaldehyde:
Fish were cut into small pieces, bones and fins were separated. The
tissue (2.0 g) was homogenized with 10 mL phosphate buffer (25 mM KH2PO4-NaOH,
pH 7.4) using a polytron homogenizer (PT 1200C, Kinematica AG, Switzerland).
The homogenates were centrifuged at 16000 x g for 20 min. (Eppendorf Centrifuge,
Model: 5415D, Germany) and supernatants were collected. Formaldehyde was
estimated in the supernatants using formaldehyde standards (25-200 μM)
in phosphate buffer pH 7.4.
The samples/standards were mixed with 34.2 mM Purpald in 480 mM HCl and
the reaction mixtures were incubated with continuous shaking for 60 min.
at room temperature. The reaction was terminated by addition of 50 μL
7.8 M KOH solution. Thereafter, the reaction mixtures were incubated further
for 10 min at room temperature and the reaction product was oxidized by
65.2 mM KOH (Johansson and Borg, 1988). The absorbance was measured at
550 nm (U best-30, JASCO, Japan). Phosphate buffer pH 7.4 was used as
Rats: A total of 28 Long Evans male rats weighing ~250
g were recruited. They were housed in an animal room at ~25°C, under
12 h dark-light cycles (light 8:00-20:00 h; dark 20:00-08:00 h) before
Oral administration of formaldehyde to rats and preparation of liver
and intestinal tissue homogenates: Of the 28 rats, 14 were orally
administered to a single dose of formaldehyde (100 mg). The remaining
14 rats were administered to a single dose of 0.50 mL phosphate buffer
pH 7.4. These rats were considered as formaldehyde un-administered control
rats. After 15 min. rats were anesthetized using pentobarbital; whole
liver and small intestine were removed. These tissues (2.0 g) were, separately
homogenized with 10 mL phosphate buffer pH 7.4 using a polytron homogenizer.
The resulting homogenates were centrifuged at 16000 x g for 20 min to
remove the unbroken tissues or cells and the supernatants were subjected
to lipid peroxide (LPO) estimation.
Preparation of liver and intestinal epithelial cell homogenates of
control rat and in vitro treatment with formaldehyde: The liver
and the small intestine of the formaldehyde un-administered control rats
were isolated. The small intestine was perfused with ice-cold phosphate
buffer pH 7.4 in waxed-Petri dish, segmented into small pieces of 7~8
cm, cleaned and epithelial cells were collected by scraping with a stainless
metallic loop. These epithelial cells and the liver tissue (2.0 g of each)
were separately homogenized with 10 mL phosphate buffer pH 7.4 using a
polytron homogenizer. Thereafter, the homogenates (50.0 μL of each)
were treated with 0, 62.5, 125, 250 and 500 μM formaldehyde for 1
h and were subjected to LPO estimation.
Estimation of LPO: LPO levels were estimated by the thiobarbituric
acid (TBA) test (Ohkawa et al., 1979) with slight modification.
In brief, the homogenates were mixed with 0.02% butyl hydroxytoluene to
inhibit spontaneous oxidation. To each 50 μL of homogenate sample,
300 μL of 8.1% sodium dodecylsulphate (SDS), 2.0 mL of 0.04% TBA
in 20% acetic acid (pH 3.5) and 500 μL of distilled water were added.
The mixtures were incubated at 95°C for 1 h. After cooling with tap
water, 1.0 mL distilled water and 2.5 mL of n-butanol-pyridine (15:1,
v/v) were added and the mixtures were shaken vigorously for about 20 min.
After centrifugation at 1000 x g for 10 min, the absorbance of the upper
organic layer was determined at 530 nm. Malonyldialdehyde levels were
calculated relative to a standard preparation of 1,1,3,3-tetraethoxypropane.
Protein assay: Homogenized tissues were heated at 80°C for
one hour in 0.2 N NaOH to solubilize the protein content. Each aliquot
was centrifuged at 2000 x g for 30 min. The supernatants were used for
protein assay according to the method of Lowry et al. (1951).
Statistical analysis: Results are expressed as mean±SEM
(Standard Error of Mean). For two-group differences, data were analyzed
by Unpaired t-test. For more than two-group differences, data were analyzed
by one-way ANOVA. ANOVA was followed by Fisher`s protected least square
differences (PLSD) for post hoc comparisons. The statistical program used
was StatView® 4.01 (MindVision Software, Abacus Concepts,
Inc., Berkeley, USA). p<0.05 was considered statistically significant.
RESULTS AND DISCUSSION
Rui fish (Labeo rohita) imported in Bangladesh from neighboring
countries are usually preserved with formalin. Therefore, it is assumed
that the imported rui fish may retain formaldehyde in their tissues. In
Bangladesh, reports are not available about the levels of formaldehyde
in these preserved fish. Most of the people of Bangladesh are not aware
about the health hazards of formaldehyde consumption with fish. This study
was conducted to examine the levels of formaldehyde in the rui fish, as
well as, to evaluate the possible detrimental effects of formaldehyde
in rat liver and small intestinal tissue.
||Formaldehyde levels in fish tissue. Results are expressed
as mean±SEM (n = 35). Unpaired t-test was performed for data
analysis. *p<0.05 was considered statistically significant
||Effect of orally administered formaldehyde on lipid
peroxidation in rat liver and small intestinal tissues. Results are
expressed as mean± SEM (n = 14). Bars with different notations
are significantly different at p<0.05. One-way ANOVA was performed
for data analysis. ANOVA was followed by Fisher`s protected least
square differences (PLSD) for post hoc comparisons
The imported rui fish and the fresh rui fish of local ponds examined
are very similar in size, shape, color and appearance. The imported rui
fish had significantly higher formaldehyde levels (~3.4 folds) than that
of the fresh rui of local ponds (Fig. 1), indicating
that excessive formalin was used for preservation of the rui fish prior
import. Formaldehyde was also present to some extent in the fresh rui
fish. The storage of fish on ice might be responsible for it, as formaldehyde
is obtained in frozen fish by means of enzymatic reactions (Bianchi et
||Dose dependent effect of in vitro formaldehyde
treatment on lipid peroxidation in rat liver tissue (A) and intestinal
epithelial cells (B). Each symbol indicates mean±SEM (n = 14).
Symbols with different notations are significantly different at p<0.05.
LPO: Lipid peroxide. One-way ANOVA was performed for data analysis.
ANOVA was followed by Fisher`s protected least square differences
(PLSD) for post hoc comparisons
In this study, the extent of tissue damage caused by formaldehyde was
also evaluated by measuring the levels of LPO. The liver and small intestinal
tissues of the orally formaldehyde-administered rats had significantly
higher LPO levels than that of the liver and intestinal tissues of the
control rats (Fig. 2). The increase in liver and in small
intestine was 4.8 vs. 3.7 folds as compared to control, indicating that
formaldehyde has considerable tissue-damaging effects in liver, as well
as, in small intestine.
The effects of in vitro formaldehyde treatment on LPO in rat liver
tissue and intestinal epithelial cells were also examined. Different concentrations
of formaldehyde (0~500 μM) were used in this purpose. Results show
that the LPO levels were dose dependently increased (p<0.05) in both
the liver tissues (Fig. 3A) and the intestinal epithelial
cells (Fig. 3B), although the effect is more prominent
in the liver tissues than in the small intestinal cells. Thus, the in
vitro effect of formaldehyde on lipid peroxidation supports the ex
vivo effect. Now, the question is, why LPO levels were increased in
liver and small intestine after formaldehyde treatment.
The production of LPO and other oxidative species like H2O2,
superoxide radicals (O••,2), hydroxyl
radicals (•OH) is an intrinsic phenomenon of normal cellular metabolism.
The oxidative species are neutralized by endogenous antioxidative enzymes
like catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase
(SOD) (Benov et al., 1990), as well as, by antioxidant substrates
like reduced glutathione (GSH) (Kidd, 1997), vitamin E (Dundar and Aslan,
2000) and other free radical scavengers. When the production of oxidative
species exceeds the endogenous protections of CAT, GPx, SOD, GSH, the
resulting damage to cellular constituents is known as oxidative stress.
After formaldehyde administration, it might have acted upon antioxidants
and reduced the activities of antioxidative enzymes and/or antioxidant
substrates; otherwise the LPO levels could have not been raised.
Formaldehyde metabolism in the body requires a number of enzymes. One
of which is formaldehyde dehydrogenase (FDH). It is present in all animal
tissues tested (Achkor et al., 2003). FDH system is active in both
the cytosol and the mitochondria. The cytosolic form is dependent on reduced
glutathione (GSH). Thus, the increased LPO levels in the liver and small
intestine after formaldehyde treatment is consistent to the fact that
GSH is exploited biochemically in order to metabolize formaldehyde.
Although, LPO levels were increased after formaldehyde treatment, it
is much higher in liver than in small intestinal tissues in both the ex
vivo and in vitro experiment. The exact reasons for
this variation are not clearly understood. The intestinal tissues serve
principally as the site for absorption of nutrients, water and both beneficial
and potentially harmful xenobiotics. In the rat, it is about 90 cm long
and its luminal surface is composed of monolayer of enterocytes with numerous
finger-like projections of villus. It is, therefore, speculated that formaldehyde
that was orally administered to the rats had limited time-exposure in
the intestine as compared to that in the liver, the ultimate first-pass
metabolic reservoir. Thus, after oral administration, the oxidative stress
of formaldehyde was lower in the intestine when compared to that in the
liver, despite its higher antioxidative defense than the former. We conclude
that after oral administration formaldehyde affects mainly the liver where
its catabolism occurs, with a concomitant increase in the extent of cell
The enzymatic catabolism of formaldehyde occurs prevalently in the liver
with the expense of antioxidants. This might be related with the more
significant effect of formaldehyde on the oxidative stress in the liver
tissues than in the small intestinal cells in in vitro experiment,
as antioxidants are exploited biochemically in order to metabolize formaldehyde.
However, further study will be required to know the actual reason.
As formaldehyde affects a diverse biochemical process of the body tissues,
people should avoid consumption of formalin-preserved fish, especially
rui fish (Labeo rohita) that come in Bangladesh from neighboring
countries. The government should take necessary measures to prevent the
use of formalin as fish preservative and also generate awareness in the
consumers about the harmful effects of formaldehyde.
This study was supported, in part, by a Grant-in-Aid from Jahangirnagar
University, Savar, Dhaka, Bangladesh [Grant No. 1234 (140), 2005].