Indian Journal of Experimental Biology Vol. 43, January 2005, pp. 96-99

Lysosomal stability in Oreochromis mossambicus (Peters) on exposure to surfactants

PC Bindu, Babu Philip* & R Vinu Chandran Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology, Kochi 682 016, India

Received 10 March 2004, revised 20 October 2004

The three commonly used surfactants viz. anionic sodium dodecyl sulfate (SDS), cationic cetyl tri methyl ammonium bromide (CT AB) and non-ionic triton X-1 00 were toxic even at sub lethal levels (I ppm for 30 days) to 0 . mossambicus. Lysosomal stability index (LSI) was lowest in triton-exposed animals in vitro. in vivo, CTAB was the most toxic. SDS, the anionic surfactant was the least toxic. The possible role of surfactant structure, critical micellar concentration (CMC) and metabolism in influencing the toxicity is discussed and mechanism of action via membrane lipid peroxidation is suggested.

Keywords: Lysosomes, Oreochromis mossambicus, Surfactant toxicity

Lysosomal damage is well-established as a bio-marker of stress in a wide range of vertebrates and invertebrates 1

·3

• The damage to the lysosomal memb­rane may be conceptualized as an increase/activation or decrease/inhibition of lysosomal hydrolases or labilizing/stal?ilizing effects due to changes in membrane permeability effected by the contami­nants4. Membrane labilization is unlikely to occur other than as a stress phenomenon, whereas elevation of activity can occur as a consequence of an increased metabolic demand, for example during gameto­genesis, rather than as a stress response5

.

Surfactant toxicity studies in fishes have revealed that liver is an important site of accumulation or metabolism of surfactants6

·7

. Hence liver lysosomes are markers of assessing the impact of surfactants on the cell membrane. Lysosomes are single membrane­bound organelles that enclose a battery of hydrolytic enzymes like acid phosphatase, beta glucuronidase, cathepsin, aryl sulfatase, etc. They have an acid environment, which is maintained by a membrane­bound Mg2

+ ATPase-dependent proton pump8• If the

lysosomal membrane is damaged or destabilized then these marker enzymes are released. Hence the assay of these enzymes can be used as an index of lysosomal membrane damage. The release of lysosomal enzymes is related to necrosis or death of

*Correspondent author Phone: +91-484-2368120 E-mail: babuphilip@cusat.ac.in

the cell or pathological stressful conditions. In the present study, the effects of three surfactants- anionic sodium dodecyl sulphate (SDS), non-ionic Triton X­I 00 and cationic cetyl trimethyl ammonium bromide (CTAB) at a sub lethal concentration of 1 ppm have been studied on the hepatic lysosomal membranes (in vitro and in vivo) of Oreochromis mossambicus.

Materials and Methods Fish were collected from Rice Research Institute,

Vytilla, Kochi, Kerala. They were fed on a commercial diet ad libitum and were acclimated in aquarium tanks for a month before the experiment. Fish specimens weighing 15 ± 3 g and 8.5 ± 0.5 em long were used for the experiments. Six fish each were exposed to a sublethal surfactant concentration of 1 ppm (1/10 of 96 hr LC50) of each of the surfactants (SDS, Triton x-100 and CTAB) in aerated fibre glass tanks. The surfactants were dissolved in tap water and diluted to obtain the required concentration of 1 ppm (APHA)9

.

The tap water used had dissolved oxygen content of 7-8 ml.L- 1

, hardness, 0 mg/L CaC03 below detectable limits, pH 7, temperature 25° ± 3°C and salinity 0 ppt. During the experimental period of 30 days, the animals were fed on the same diet so as to avoid the effects of starvation on normal physiological process and antioxidant status. Water in the experimental tanks was replaced every 48 hr with water containing fresh surfactant so as to avoid any possible degradation of the surfactant. A control group of six

BINDU et al. : LYSOSOMAL STABILITY IN OREOCHROMIS 97

fishes was also maintained without any surfactant. Feeding was stopped 24 hr before sacrificing the animals. The fish were sacrificed by pithing and the liver tissue was dissected out, washed in ice-cold isotonic sucrose (0.33 M), blotted dry and weighed.

Activity of acid phosphatase (ACP) in various sub cellular fractions of the liver tissue--l..i ver was homogenized in ice-cold isotonic sucrose at 0°C. The homogenate was centrifuged at 600 g in a high speed refrigerated centrifuge. The sediment of nuclei, unbroken cells and plasma membrane (nuclear fraction) was separated. The supernatant was again centrifuged at 15000 g for 30 min. The 15000 g sediment (lysosomal fraction) and the nuclear fraction were resuspended in 0.1 M citrate buffer, pH 4.8 containing 0.2% Brij-35 (mild non-ionic detergent used commonly for membrane solubilisation). The 15000 g supernatant (soluble fraction) was diluted with an equal volume of double strength buffer (0.2 M, pH 4.8). Activity of acid phosphatase was determined in all these fractions 10

.

Rate of release of acid phosphatase (ACP) from lysosomal fraction or lysosomal enzyme release assay (LERA)-(i) In vitro studies: Liver from control animals was

homogenized in isotonic sucrose and the lysosomal fraction was obtained as above. The lysosomal pellet was resuspended in sucrose. A definite volume of this suspension was incubated at room temperature and aliquots were withdrawn at time intervals of 0, 15 and 30 min. The retrieved fractions were stored at 0°C (control). In order to study the effects of surfactants on the lysosomal membrane, a definite volume of the lysosomal suspension (test) was incubated in presence of each of the surfactants such that the final concentration of surfactant was 1 ppm. Here also aliquots were withdrawn at 0, 15 and 30 min. Both control and test aliquots were centrifuged at

15000 g for 30 min to separate the unbroken lysosomes and acid phosphatase activity released into the supernatant was determined. Total lysosomal activity was estimated after adding citrate buffer containing 0.2% Brij-35. For evaluation of the effects of surfactants on enzyme release, the possible effect of surfactant (inhibition or activation) was also taken into account.

(ii) In vivo studies: The hepatic tissue from the control fish and those exposed to the surfactants for 30 days were homogenized separately and centrifuged to obtain the lysosomal fraction as described earlier. The rate of release of acid phosphatase was measured at time intervals of 0, 15, and 30 min by withdrawing definite aliquots (0.2 ml) of the lysosomal suspension as described in in vitro study.

Determination of enzyme activity11--p-nitro phenol (400 mg%; 0.5 ml) was mixed with an equal volume of 0.1 M citrate buffer (pH 4.8). The enzyme was added and incubated at room temperature for 30 min. The reaction was stopped by adding 4 ml of 0.1 N NaOH. The absorbance of the solution was measured at 410 nm in UV-visible spectrophotometer (Hitachi). The amount of p-nitro phenol liberated by the enzyme per hour per mg protein gives the specific activity. Protein was estimated by the method of Lowry et al 12

•

Statistical analysis-Statistical analysis of the results obtained was done by ANOV A followed by Least Significant Difference (LSD) 13

•

Results Subcellular act!Vlty of acid phosphatase

(ACP}-Subcellular activity of acid phosphatase is given in Table 1. One way analysis of variance (ANOV A) revealed an overall significant change (P<O.OOI) in ACP activity in nuclear (F=157.82), lysosomal (F=327.06) and soluble fractions (F=156)

Table I --subcellular activity of acid phosphatase in O.mossambicus exposed to I ppm surfactants in vivo

[Values expressed as mg p-nitrophenolliberated/hr/mg protein in each fraction are mean ± SD of 6 separate experiments] '

Groups Nuclear activity Lysosomal activity Soluble activity

Control 5.789 ± 0.92 11.99 ± 0.91 SDS dosed 10.55 ± 0.52 *3 8.89 ± 0.83 *ac Triton dosed 9.09 ± 0.47 *" 2.48 ± 0.82 *ab CT AB dosed 8.87 ± 0.43 *c 6.41 ± 0.88 *be *P<O.OO!. Significant differences between SDS and CT ABare represented as a, band c respectively.

6.1 ± 0.85 7.18 ± 0.9*ac · 12.01 ± 0.89*ab 10.65 ± 0.88*bc

Triton, Triton and

Lysosomal Stability Index (LSI) 1.965 1.23 0.207 0.591

CT AB and ~DS and

98 INDIAN J EXP BIOL, JANUARY 2005

of experimental animals. Subsequent comparisons between different groups were done by LSD.

There was a significant increase in nuclear and soluble ACP in all the surfactant-treated groups when compared with the control (P<0.001). On the other hand a significant decrease was noted in lysosomal ACP of surfactant-exposed fish when compared to the control (P<O.OOl). Comparison between surfactants revealed no significant differences in the nuclear ACP in animals exposed to Triton x-100 and CTAB, but there were significant differences (P<O.OO I) between the surfactants with respect to the lysosomal and soluble ACP activities. The ratio of ACP activity in lysosomal fraction to that in the soluble fraction or Lysosomal Stability Index (LSI) was the lowest for animals exposed to Triton x-100 (0.207). It was followed by CT AB, which had an LSI of 0.591, and the anionic SDS had an LSI of 1.23. LSI for control group was 1.965.

LERA (in vitro studies}--The time-dependent release of ACP from lysosomes in vitro and in vivo is given in Table 2. There was an increased release of ACP from the lysosomes of the surfactant-exposed fish with time when compared to control. Two way ANOVA (comparing ACP release with time) revealed an overall significant change (P<0.05) in the release of ACP from lysosomes of the experimental groups when compared to the control. Subsequent LSD analysis reflected significant differences (F=l9.82, P<0.05) in the release of ACP with time, i.e., there was a progressive increase in ACP release from surfactant-treated lysosomes from 0 min to 30 min.

LERA (in vivo studies~Two way ANOVA on in vivo studies showed that there was no significant difference in the ACP release with time (Table 2). But the surfactant exposure was found to induce

an overall significant change (F=6.5, P<0.05). Comparison by LSD analysis revealed that only CT AB exposure caused a significant rise in ACP release (P<0.05) when compared to the control. This observation, different from that in in vitro studies, points to the fact that in vivo metabolism of the surfactant may be responsible for the change in the pattern of ACP release from lysosomes in vivo.

Discussion The lysosomal ACP in surfactant-dosed fish was

lower than that of control but the soluble activity was highly increased. This indicates damage to the lysosomal membrane on exposure to surfactants. The interaction of surfactants with the lysosomal membrane involves two aspects. First there are hydrophobic as well as hydrophilic interactions with membrane lipids and proteins, and the second is that the interaction with the membrane lipids causes peroxidation. The concentration of the surfactant plays a major role in the interactions. The membrane is rendered leaky at much lower concentration than required for complete solubilization. Also this concentration increases with an increase in critical micellar concentration (CMC) of the surfactant 14

•

Thus a smfactant of low CMC causes release of cell/cytoplasmic protein by intercalating with the membrane bilayer without much solubilisa­tion. Thus the decreasing order of toxicity is Triton x-100> CTAB > SDS .

Triton x-I 00, the non-ionic surfactant, and CT AB the cationic surfactant have comparatively much lower CMC than SDS, the anionic surfactant. This may have caused increased release of acid phosphatase into the soluble fraction in presence of Triton and CT AB. This is evident from an LSI of

Table 2-Lysosomal enzyme release assay

Time (min)

0

15

30

[Values expressed as mg p-nitrophenol Iiberated/hr/mg protein in each fraction are mean± SD of 6 separate experiments]

Control SDS Triton

A 1.06 ± 0.21 0.957 ±0.03 1.77 ± 0.06 B 0.702 ±0.23 0.798 ± 0.02 0.922 ± 0.04

A 1.135 ± 0.02 1.063 ± 0.01 *ac 1.915 ± 0.05*" B 0.936 ±0.04 0.979 ± 0.11 c 1.064 ± 0.01 b

A 1.21 ± 0.01 1.276 ± 0.02*ac 2.27 ± 0.02*ac B 1.035 ± 0.02 1.138 ± 0.08 c 0.993 ± 0.05b

A = in vitro, B = in vivo

CTAB

1.01 ± 0.05 0.8 ± 0.095

1.17 ± 0.0 I *c 1.596 ± o.o5 · be

1.33 ± 0.02*c 1.862 ± 0.04. be

*?<0.05. Significant differences between SDS and Triton, Triton and CTAB and SDS and CTAB are represented as a, band c respectively.

BINDU et al.: LYSOSOMAL STABILITY IN OREOCHROMIS 99

0.207 for Triton, 0.591 for CTAB and 1.23 for SDS, compared .to the control LSI of 1.965.

Results of lysosomal release assay (LERA) in vitro revealed a significant increase in ACP release with time. This demonstrates a direct interaction of surfactants with the lysosomal membrane. The effects of Triton were significantly different from that of SDS and CT AB, which had similar labilizing effects. But in vivo studies on LERA presented a different result. Here the cationic CT AB was the most labilising when compared to the control. Triton was next to CT AB in toxic effects and SDS was the least toxic. It could be inferred that metabolism of the surfactant in vivo leads to a change in toxicity pattern. It is well documented that CT AB being very non polar is not metabolized in fish. S'J CT AB is the most toxic. SDS is metabolized via omega and subsequent ,8-oxidation and is the least toxic. Triton x-100 is metabolized as glutathione conjugates which depletes glutathione in the animal and renders it more susceptible to the deleterious effects of lipid peroxidation 15

• Damage via lipid peroxidation to lysosomal membranes cannot be overruled. Though lysosomal membrane has only 25% lipids, it is still susceptible to the deleterious effects of peroxidation. The peroxidative potential of the surfactants used in the present study has been proved 16

• Thus it may be concluded that surfactant interactions with the cell membrane lipids and proteins as well as lipid peroxidation are key factors in the damage caused to the lysosomal membrane in vitro whereas metabolism of the surfactant is a key factor in deciding in vivo toxicity .

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