Arch. Environ. Contam. Toxicol. 52, 355–362 (2007) DOI: 10.1007/s00244-006-0056-9
Environmental Contamination of Chrysotile Asbestos and Its Toxic Effects on Antioxidative System of Lemna gibba A. K. Trivedi,1 I. Ahmad,2 M. S. Musthapa,2 F. A. Ansari2 1 Department of Life Science, Amity School of Engineering, Amity University Uttar Pradesh, Super Express Highway, Sector – 125, Noida – 201 303, India 2 Fibre Toxicology Division, Industrial Toxicology Research Centre, M. G. Marg, P. B. No. 80, Lucknow – 226 001, India
Received: 17 March 2006 /Accepted: 20 November 2006
Abstract. Asbestos was monitored in various plant samples around an asbestos cement factory. Asbestos residue was found on the surface of all plant samples monitored. Based on asbestos concentration found in different plant samples during monitoring and on the property of asbestos to cause reactive oxygen species-mediated oxidative stress in animal models, laboratory experiments were conducted to assess the toxicity of chrysotile asbestos on an aquatic macrophyte, duckweed (Lemna gibba.). L. gibba plants were exposed to four concentrations (0.5, 1.0, 2.0, and 5.0 lg/mL) of chrysotile asbestos under laboratory conditions, and alterations in the glutathione and ascorbate antioxidative system were estimated at postexposure days 7, 14, 21, and 28 in order to assess changes in their level as suitable biomarkers of chrysotile contamination. Chrysotile exposure caused a decrease in total and reduced glutathione and an enhancement in the oxidized glutathione as well as the reduced/oxidized glutathione ratio. An increase in ascorbate pool size, and reduced as well as oxidized ascorbate was found to be accompanied by a decrease in the ratio of reduced/oxidized ascorbate. Alteration in the glutathione and ascorbate level might be considered as a biomarker of exposure to an unsafe environment because these are essential compounds of the general antioxidative strategy to overcome oxidative stress due to environmental constraints. Because an increase in the oxidation rate of antioxidants weakens cellular defenses and indicates a precarious state, they could constitute indicators of toxicity.
Asbestos fibers are divided into two classes, chrysotile and amphibole, on the basis of their crystal structure (Light and Wei 1977). Crysotile is a fibrous, hydrated magnesium silicate mineral [Mg3Si2O5(OH)4] that is used in approximately 3000 commercial products (Ramanathan and Subramanian 2001). In India, several states have many asbestos industries, of which 60% are in operation, and production is about 2000 tons per month (Ramanathan and Subramanian 2001). Air pollution Correspondence to: A. K. Trivedi; email:
[email protected]
levels of asbestos were reported to be elevated in the areas surrounded by asbestos industries (IPCS 1998). During factory operation, asbestos fibers are released in the environment. Therefore, monitoring and analysis of biotic and abiotic samples in the nearby ecosystem can address many questions about source, distribution, partitioning, and transport of asbestos. In natural condition, chrysotile fibers can be transported by wind and water (IPCS 1998). Natural fresh water is an important receptor of many toxic substances released by industrial, agricultural, and domestic activities (Kumari et al. 2001). The rapid decline of water quality resulting from industrial waste water during recent years has become one of the greatest environmental problems (Danilov and Ekelund 2001). Various health effects in humans and laboratory mammals have been documented about chrysotile fiber exposure (Hauptman et al. 2002). However, potential ecological impacts of this material have largely been ignored (NIPHEP 1989). There have been some attempts to show the interconnections between human and ecosystem health (Di Giulio and Monosson 1996). Efforts to develop an integrated approach to ecological and human health risk assessment are desirable for efficacy, cost effectiveness, and to garner public support (Harvey et al. 1995; Burger and Gochfeld 2001). Furthermore, one of the great generalizations of cell biology is that the cells of higher organisms, be they from plants or animals, are fundamentally similar (Prescott 1982). Chrysotile fibers carry a positive surface charge at pH < 11.8 (Speil and Leinweber 1969). These charged fibers presumably would be attracted to negatively charged protein groups in cell membranes. The chrysotile fiber would then be surrounded by proteins and submerge into the cell. This series of events could be a mechanism by which fibers could gain entry into the cell (Harington et al. 1975). Moreover, a common effect of many pollutants is to generate free radicals and reduced forms of oxygen that may damage cellular components such as lipids, proteins, or DNA (Foyer et al. 1997). The importance of reactive oxygen species (ROS) in contributing asbestos-associated cytotoxicity has been reported by several studies (Kamp et al. 1992). Several investigators have shown that fiber-cell interaction is not necessary for production of ROS. For example, chrysotile and crocidolite asbestos in
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cell-free solutions of water or physiological saline spontaneously generate superoxide (O.2) or hydroxyl radical (OH.)) (Ebenhardt et al. 1985). Duckweed (Lemna) is an important food species for aquatic herbivores, a good dietary supplement and nutrient source (Oron et al. 1985) for humans (Majid et al. 1984), livestock and fish (Lehman et al. 1981), and is used as a good fertilizer supplement (Mabagwu and Adeniji 1988) and also as an indicator of water pollution (Nasu and Kugimoto 1981). It is widely recommended for aquatic toxicity studies (U.S. EPA 1985). The selection of exposure concentration is based on the range of fibers found during monitoring (Rahman et al. 2001) and our previous study (Trivedi et al. 2004). In this study, we report environmental contamination of chrysotile asbestos in the vicinity of an asbestos cement factory and its toxic effects on the glutathione-ascorbate antioxidative system of a primary producer, Lemna gibba.
Materials and Methods Asbestos Fiber Analysis Asbestos analysis in different samples was carried out following the method of APHA et al. (1998), U.S. EPA (1993), and IS (1986). Plant samples were collected at different locations around an asbestos cement factory, which is located at Mohanlalganj about 25 km from Lucknow, U.P. (India). Twenty-one species of terrestrial plants present surrounding the factory were undertaken for the study. For counting fibers on the leaf surface of different plants, 10 samples of each terrestrial plant were collected quarterly, and 1.0-g leaves from each sample were washed with 100 mL of distilled water. The water samples from leaf washing were filtered through a Millipore membrane filter paper with pore diameter 0.8 lm (Cat No. AABP 04700, Millipore Corp., Bedford, MA), which retains asbestos fibers present in the samples, and were subsequently transferred onto a slide and made transparent by the addition of 200–300 lL standard immersion oil (Olympus Japan). Transparent slides were air dried and used for asbestos analysis by the phase-contrast polarized microscopic (PCM) method (IS 1986). The length of asbestos fibers was measured in the range of 20 lm, and the relative count of fibers was also estimated in the original material. Data presented are an average of three-successive-year sampling.
Collection and Culture of Lemna gibba To study toxic effects of chrysotile asbestos on L. gibba, plants were collected from the natural habitat in an aquatic body, washed axenically, and maintained in Hoagland medium (U.S. EPA 1975) in the laboratory under light and dark period of 16/8/day and controlled humidity (60%). The young plants of the third generation were transferred to sterilized Petri dishes and used for experiment. Chrysotile fibers of size 20 lm compared to small fibers. This situation might be due to the fact that small fibers travel a greater distance through the air as compared to longer ones. According to Toxic Release Inventory (TRI) in 1999, the total releases of asbestos to the environment (including air, water, and soil) from 87 facilities were 13.6 million pounds (TRI 99 2001).
Effects on Glutathione Content It is currently assumed that the negative effect of the various environmental stresses is at least partially due to the generation of active oxygen species (AOS) and/or the inhibition of the system, which defends against them (Shalata and Tal 1998). The importance of AOS in contributing to asbestos-associated cytotoxicity has been indicated by several studies (Mossman et al. 1986; Kamp et al. 1992). In addition to alteration in the activity of antioxidant enzymes, change in the antioxidant pool size is an adaptation to stress and a defense process. Figure 1 displays depletion of total glutathione content. In plants exposed to 0.5, 1.0, 2.0, and 5.0 lg/frond chrysotile, the total glutathione pool size decreased by 9.92%, 13.40%, 16.51%, and 21.31%, respectively, at postexposure day 7; at postexposure day 28 the decrease was 46.49%, 55.63%, 59.30%, and 69.87%, respectively. Depletion in total glutathione content might be due to the inability of plants to overcome stress generated by chrysotile exposure. The tripeptide glutathione (GSH), an antioxidant, exerts a number of functions in plants (Paranhos et al. 1999). It is a biomarker of exposure to an unsafe environment. In conformity with total glutathione content, depletion of 5.14%, 8.14%, 11.33%, and 14.40% at postexposure day 7 and 38.67%, 56.67%, 58.33%, and 70.67% at postexposure day 28 was found in the profile of GSH, in plants exposed to 0.5, 1.0, 2.0, and 5.0 lg/frond chrysotile, respectively (Fig. 2). GSH is one of the main reducing agents
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in the cell, and it recycles ascorbic acid from its oxidized to its reduced form. A gradual and steady decrease found in GSH content indicates weakening of the internal defense mechanism in exposed plants. With increase in concentration and exposure time, GSSG contents were increased, which is in contrast to the effect on total glutathione and GSH. This might be due to enhanced oxidation of glutathione in exposed plants. Chrysotile exposure caused an increase of 14.10%, 28.71%, 33.33%, and 43.85% at postexposure day 7 and 85.10, 97.18, 103.61, and 117.35% at postexposure day 28 in 0.5, 1.0, 2.0, and 5.0 lg/frond of chrysotile-exposed plants, respectively (Fig. 3). Increase in glutathione oxidation resulted in increased GSSG content and consequent decrease in GSH/GSSG ratio. GSH/GSSG ratio in control plants was 19.37, which decreased to 18.51, 17.01, 16.82, and 16.25 at postexposure day 7 and 8.85, 8.76, 8.61, and 8.52 at postexposure day 28 in 0.5, 1.0, 2.0, and 5.0 lg/frond of chrysotile-exposed plants, respectively (Fig. 4). Increased oxidation of glutathione affects the defense mechanism because a major function of reduced glutathione in the protection of cells against the toxic effects of free radicals is to keep free-radical scavenging ascorbate in its reduced, hence active form by involvement in the ascorbateglutathione cycle (Zhang and Kirkham 1996). Furthermore, it is involved in the detoxification of the xenobiotics as a substrate for the enzyme glutathione S-transferase (Marrs 1996). It also participates in the protection against heavy metals as a precursor of the phytochelatins, which are metal-binding peptides in plants (Rauser 1995). Similarly to glutathione, ascorbate was predominantly present in its reduced state (AsA). As a response to chrysotile exposure, an increase of total ascorbate content was found. At postexposure days 7 and 28, an increase of 8.71%, 9.18%, 16.18%, 21.57% and 29.18%, 38.50%, 59.46%, 79.41% in 0.5, 1.0, 2.0, and 5.0 lg/frond of chrysotile-exposed plants, respectively, was found (Fig. 5). This might be due to the stimulation of antioxidant metabolism by increases in ROS, although basal developmental factors may also play a decisive role (Donahue et al. 1997). Figure 6 shows an enhancement of AsA level until postexposure day 21. At postexposure day 21, an increase of 18.82%, 22.58%, 42.47%, and 56.99% was found in 0.5, 1.0, 2.0, and 5.0 lg/frond of chrysotile-exposed plants, respectively. At a later stage (i.e., postexposure day 28), a slight decrease in AsA level was found as compared to the AsA level at postexposure day 21. The general aspect of the diagram showing variation of DAsA after chrysotile exposure was nearly identical to the AsA one, with slight differences (Fig. 7). A concentrationdependent increase of DAsA content was observed up to postexposure day 21 and a slight decrease at postexposure day 28. At postexposure day 21, the magnitude of increase was 32.73%, 34.64%, 37.18%, 40.27%, which was only 30.36%, 31.18%, 33.09%, 35.54% at postexposure day 28 in plants exposed to chrysotile at the rate of 0.5, 1.0, 2.0, and 5.0 lg/ frond. In the control plants, the AsA/DAsA ratio was 3.38, after chrysotile exposure this ratio gradually decreased to a level of 2.03 in 5.0 lg/frond chrysotile exposure at postexposure day 28 (Fig. 8). This decrease in AsA/DAsA ratio may cause oxidative damage, because when plants are subjected to environmental stress, the balance between the production of ROS and quenching activity of antioxidants may be upset and oxidative
A. K. Trivedi et al.
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Table 1. Asbestos burden on the surface of different plants around asbestos cement factory % Fibers (lengthwise) S. no.
Name of the plant
Total fibers/g dw
20 lm
1
Luffa acutangula (Nasdar Torai) Oryza sativa (Dhan) Azadirachta indica (Neem) Madhuca indica (Mahua) Chenopodium album (Bathua) Hibiscus rosa-sinensis (Gurhal) Musa paradisiacal (Banana) Cyanodon dactyloI (Doob grass) Cajanus cajan ( Arhar) Mangifer Indica (Am) Acaci nilotica (Babool) Syzygium cumuni (Jamun) Solanum tuberosum (Alu) Eucalyptus globules (Eucalyptus) Rosa indica (Gulab) Carica papaya (Papita) Brassica oleracea (Bandgobhi) Zea mays (Maize) Psidium gujava (Amrud) Lagenaria Siceraria (Lauki) Ziziphus maurintiana (Ber)
153
11.8% (18)
13.1% (20)
75.16% (115)
149
10.1% (15) 7.7% (11)
15.4% (23) 15.5% (22)
74.49% (111) 76.76% (109)
140
8.6% (12)
11.4% (16)
80.0% (112)
166
9.0% (15)
15.7% (26)
75.3% (125)
155
12.9% (20) 11.1% (18)
16.1% (25) 17.9% (29)
70.96% (110) 70.98% (115)
174
11.5% (20)
17.8% (31)
70.68% (123)
123
13.0% (16) 15.0% (12)
12.1% (15) 13.7% (11)
74.79% (92) 71.25% (57)
21.5% (19) 15.6% (15)
19.3% (17) 21.8% (21)
59.09% (52) 62.50% (60)
69
11.6% (8)
21.7% (15)
66.66% (46)
99
17.1% (17)
18.2% (18)
64.64% (64)
95
12.6% (12) 10.6% (7) 8.1% (5)
17.9% (17) 22.7% (15) 27.4% (17)
69.47% (66) 66.66% (44) 64.51% (40)
16.3% (11) 13.4% (10) 14.0% (8)
18.1% (14) 22.7% (17) 24.6% (14)
67.53% (52) 64.0% (48) 61.4% (35)
12.1% (8)
18.1% (12)
69.69% (46)
2 3
4
5
6 7
8
9 10
11 12
13
14
15 16 17
18 19 20
21
142
162
80
88 96
66 62
77 75 57
66
Figure within parentheses indicates number of fibers out of total number.
damage may result (Dhindsa and Matowe 1981; Spychalla and Desborough 1990; Cakmak and Marschner 1992). The increase of ROS seems to occur as a response to all environ-
mental stresses, including drought (Jagtap and Bhargava 1995), salt (Gossett et al. 1994a, 1994b, 1996), extreme temperatures (Mahan 1994; Prasad et al. 1994), nutrient deficiency
359
Chrysotile Asbestos Effects on Lemna gibba
100
0.5 µg
90
1.0 µg
*
80
2.0 µg
*
*
Percent control
70
*
*
5.0 µg
*
60
*
50
*
*
* *
40
**
30 20 10 0 7 day
14 day 21 day Post-exposure tim e
28 day
120
Fig. 1. Total glutathione content in Lemna gibba. Values presented are mean € SE expressed as percent of respective controls. Control values were 78.90 € 3.43, 77.65 € 3.22, 78.03 € 43.76, 79.55 € 3.66 lmol/g FW at postexposure day 7, 14, 21, and 28, respectively. *p £ 0.05, *p £ 0.01
0.5 µg 1.0 µg 2.0 µg
Precent of total value
100
5.0 µg
*
80
* * *
* * *
60
* *
* *
40
* 20 0 7 day
14 day
21 day
28 day
Post-exposure time
Fig. 2. GSH content in Lemna gibba. Values presented are mean € SE expressed as percent of respective controls. Control values were 75.49 € 3.13, 75.12 € 3.53, 74.40 € 3.34, 74.49 € 2.81 lmol/g FW at postexposure day 7, 14, 21, and 28, respectively. *p £ 0.05
250
Percent control
200
*
150
*
*
* *
*
*
*
*
* *
0 21 day
Post-exposure time
(Iturbe-Ormaetxe et al. 1995), and air pollution (Badiani et al. 1993). The resistance to environmental stress may therefore depend, at least partially, on the inhibition of ROS production or the enhancement of antioxidant levels. Furthermore, at the genetic level, expression of genes encoding antioxidant enzymes have been shown to change in some plants when subjected to environmental conditions such as chilling stress (Pinhero et al. 1997); light intensity and type (Willekens et al. 1994a, 1994b); salt stress (Fadzilla et al. 1997); pathogens (Williamson and Scandalios 1992); herbicides (Donahue et al. 1997); and several gaseous pollutants (Price et al. 1990; Sharma and Davis 1995; Torsethaugen et al. 1997). These reports suggest that overproduction of antioxidant enzymes could lead to tolerance to damage by chrysotile
1.0 µg
5.0 µg
50
14 day
0.5 µg
2.0 µg
100
7 day
**
28 day
Fig. 3. GSSG content in Lemna gibba. Values presented are mean € SE expressed as percent of respective controls. Control values were 3.90 € 0.53, 4.02 € 0.66, 3.99 € 0.67, 4.06 € 0.59 lmol/g FW at postexposure day 7, 14, 21, and 28, respectively. *p £ 0.05, **p £ 0.01
exposure. As main antioxidants, the increase of glutathione and/or ascorbate pool sizes after exposure to chrysotile is an attempt to overload the stress imposed by chrysotile asbestos. This could characterize the resistance stage, in which defense and adaptation metabolisms are more stimulated, leading to a hardening of plants by establishing a new physiological standard (Lichtenthaler 1996). However, when plants are unable to cope with the stress condition, a reduction in antioxidant pool size might be found. Because they were involved in the general defense strategy to cope with ROS and reactive compounds, induction of antioxidants reflected exposure to an unsafe environment responsible for the increase of harmful compounds. Thus, one could consider this increase of antioxidant pools as a potential biomarker of exposure that can be suitable
A. K. Trivedi et al.
360
25 Control 0.5 µg 20
1.0 µg 2.0 µg 5.0 µg
Ratio
15
*
10
*
* * * *
5
0 7 day
14 day
21 day
28 day
Fig. 4. GSH/GSSG ratio in Lemna gibba. Values presented are mean € SE. *p £ 0.05
Post-exposure time
200
*
180 Percent control
160
*
*
140
*
* *
120
*
*
*
*
0.5 µg 1.0 µg 2.0 µg 5.0 µg
*
100 80 60
Fig. 5. Total ascorbate content in Lemna gibba. Values presented are mean € SE expressed as percent of respective controls. Control values were 2.41 € 0.35, 2.52 € 0.45, 2.54 € 0.67, 2.43 € 0.72 lmol/g FW at post-exposure day 7, 14, 21, and 28, respectively. *p £ 0.05
40 20 0 7 day
14 day
21 day
28 day
Post-exposure time
0.5 µg
180
1.0 µg
*
160
*
Percent control
140
2.0 µg 5.0 µg
120 100 80 60 40 20 0
7 day
14 day
21 day
28 day
Post-exposure time
for monitoring water quality and pollution (Teisseire and Vernet 2000). Results obtained from monitoring and laboratory study suggested that antioxidant content in L. gibba might serve as a suitable biomarker of exposure to environmental contamination of asbestos and an early indicator of toxicity. Further research is required to validate their use under field conditions.
Acknowledgments. Authors are thankful to Dr. P. K. Seth, Director, Industrial Toxicology Research Centre, Lucknow for his
Fig. 6. AsA content in Lemna gibba. Values presented are mean € SE expressed as percent of respective controls. Control values were 1.86 € 0.35, 1.82 € 0.47, 1.88 € 0.47, 1.80 € 0.42 lmol/g FW at postexposure day 7, 14, 21, and 28, respectively. *p £ 0.05
keen interest in the study and Mohd. M. Ashquin for his skillful technical assistance. Financial assistance from Ministry of Environment and Forests (Govt. of India), New Delhi is also gratefully acknowledged.
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361
Chrysotile Asbestos Effects on Lemna gibba
180 0.5 µg 160
* *
Percent control
140
*
120
1.0 µg
* * * *
* * * *
2.0 µg 5.0 µg
100 80 60
Fig. 7. DAsA content in Lemna gibba. Values presented are mean € SE expressed as percent of respective controls. Control values were 0.55 € 0.09, 0.52 € 0.08, 0.57 € 0.07, 0.53 € 0.07 lmol/g FW at postexposure day 7, 14, 21, and 28, respectively. *p £ 0.05
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3.5 3
Ratio
2.5
*
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* *
1.5 1 0.5 0 7 day
14 day
21 day Post-exposure time
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