Title
Name
Name of Advisor
: Using frogs as biosentinel species for contamination with
melanomacrophages as histological indicator
: Maulida Aisyah Khairunnisa makhairunnisa@gmail.com
: Assist.Prof.Dr.Noppadon Kitana noppadon.k@chula.ac.th
Dr.Jirarach Kitana jirarach@yahoo.com
1.
Introduction
Contamination should be awared in our environment. Agricultural practices have been use irrigation and chemical pest control to maximize profit and production.Among these chemicals in Nan Province, more than 92.04% was herbicides (1,172,700 kg), followed by
4.44% (56,600 kg) of fungicides and 3.52% (44,800 kg) of insecticides .In this study, the example of herbicide are atrazine,glyphosate,and paraquat which are being contamination in
Thailand and the other kind of contamination in Thailand is heavy metal such as cadmium(Chanpong, 2008). The presence of them where it should not be or at concentrations because it is toxic can cause some effects of contamination. Atrazine can turn male frogs into females.Atrazine is known endocrine-disrupting chemical that could interferes with the production of the sex hormone estrogen(Hayes et al., 2010) .The example of cadmium effect is Itai-itai disease( Nishijo et al., 2017 ) .It is generally portrayed in Japan that causes weakening of bone structures. The example of paraquat effect is the skin developed erythema followed by blistering and hemorrhaging hemorrhagic diabrosis( Zhou et al ., 2013 ).
Glyphosate can cause the kidney cancer( Xie et al ., 2016 ) . Overall, The contaminants could suppress immune defenses and magnify the effects of disease. Immune responses could be weaken by contaminants potentially leading to increased risk of parasitism and disease.
Sentinel species is an organism that used to determine the ecological risk and monitoring the environment by providing advance warning of danger(National Research
Council, 1991). Some animals can act as sentinels because they might be more susceptible or have greater chance of exposure to a particular hazard than humans in the same environment. Amphibians are regarded as one of a good sentinel species for environmental contamination. Their life cycle in both aquatic and terrestrial habitats and their semipermeable ventral skin ,and unshelled egg,are making them susceptible to pesticide exposure through several routes . Amphibian size is generally suitable for enumeration and capture, and its population is relatively large. Furthermore, as a member of vertebrates, the measurable effect occurred in amphibian could also occur to other vertebrates including human Morphological studies of amphibian can thus be used to evaluate long-term impact of pesticide contamination on vertebrates( Roy, 2002 ) .
In this study ,Two frog species were chosen. Microhyla fissipes is living in the cadmium contaminated areas of Mae Sot District, Tak Province, Thailand. This frog is a candidate sentinel species because it is a small vertebrate commonly found in this area, They could be

exposed to cadmium contamination throughout their lifespan via skin penetration and dietary intake. Moreover, among small vertebrates in the area, this species is abundant and can be found throughout the wet season. The other species is Fejervarya limnocharis (rice frog) . This frog is a candidate sentinel species because they are widely distributed, tolerant to a broad range of habitats, and their population is presumably large and appears to be stable at present. Its size is relatively small with the total length of approximately 42-46 millimeters
(mm). Rice frog was used for human consumption at the higher rate than other amphibians.It also serves as an important link in the natural food web because it is a major food for several kinds of snakes. It inhabits most open wet habitat types, including river floodplains, wet agriculture areas such as rice fields, ditches, marshes, parks, gardens and other habitats,Its size is generally suitable for enumeration and capture.
Environmental contamination may cause adverse responses in animals at different structural levels, including cells, tissues, and organs .In this study, liver was observed for contamination effect because liver has hepatocytes and kupffer cells (KCs) that plays a fundamental role in biotransformation process. Therefore, the liver of ectothermic vertebrates, such as amphibian, is the most suitable organ for evaluating response against environmental pollutants These effects depend on a variety of factors, such as the type of contaminant and its concentration, the rate of exposure, and the susceptibility of the organisms. Kupffer cells in the liver of ectotherms contain melanic pigment in cytoplasm, making them known as melanomacrophages.
Melanomacrophage centers (MMCs) or macrophage aggregates (MAs) or macromelanophage center are aggregates of highly pigmented phagocytes. Melanomacrophages, including hemosiderin, lipofuscin and/ or melanin could reflect possible pathological processes and tissue destruction. Hemosiderin, an iron-containing pigment, is a breakdown product of red blood cells. Lipofuscin or ceroid is formed from the oxidative polymerization of polyunsaturated fatty acids and protein Their function has been associated with 1) inflammatory and humoral responses; 2) cell and compound storage, destruction, and detoxification of exogenous and endogenous waste products; and 3) iron recycling. MMCs frequency and pigment composition in fishes are dependent on species, health status and nutrition and age. This study has aimed to determine the contamination effect on melanomacrophage and melanomacrophage center (MMC) of Microhyla fissipes in two population (low cadmium contaminated site and high cadmium contaminated site and to determine the contamination effect on melanomacrophage and melanomacrophage center
(MMC) of Fejervarya limnocharis in different degree of herbicide utilization at Nan
Province,Thailand.
2.
Methods a.
Study Sites

(a) (b)
Fig 1. (a). Cadmium contamination area.(b). Herbicide contamination area
Two potentially cadmium-contaminated sites in Mae Sot District, Tak Province,
Thailand were chosen in this study. Mae Tao creek (High Cadmium Habitat) is a major irrigation stream for agricultural activities pass through a zinc mining area where cadmium is believed to be released to the aquatic environment due to mining activities. Huay Luek creek
(Low Cadmium Habitat) is located 10 kilometers northeast of Mae Tao creek is on a different tributary basin and not on the path of cadmium-contaminated sediment plume.
The herbicide study sites located in Wiang Sa District, Nan Province included contaminated site which is an agricultural area with intensive herbicide usage located in San
Subdistrict and reference site which is an organic agricultural area with no history of herbicide usage for almost 10 years located in Lainan Subdistrict
II.
Animal Collection a.
Cadmium Contamination Analysis
Frog’s body sample from cadmium contamination site was oven-dried at 80 o C overnight. Combination of concentrated nitric acid and hydrogen peroxide in the ratio of 7:1 was added to the sample in microwave vessels. The sample was then microwaved (980 kW) at 200 o C. The digested tissue sample was cooled to room temperature before filtered.
Distilled water was added to dilute the sample volume. Sample blank (distilled water) was also added to the same microwave procedure. Tissue sample was analyzed for cadmium content with graphite furnace atomic absorption spectroscopy in the unit of mg/L. The concentration of cadmium in the tissues was then calculated into the unit of mg/kg dry weight. b.
Histology and Melanomacrophage Counting
Liver organ specimens from both studies were fixed in 10% neutral buffered formalin.Liver tissues were processed through paraffin method (Humason, 1979), cut at 5 µm

with a rotary microtome (Leica RM2165) and stained with Delafield’s hematoxylin and eosin
(H&E; Humason, 1979). Number of melanomacrophageand MMC in liver were counted using oculargrid (Olympus eyepiece micrometer) under 100x magnification of a light microscope
(Olympus CH-2) c.
Hepatosomatic Index
Data of the body and liver from frog in cadmium contaminated site were recorded and used to calculate the hepatosomatic index(HSI) with the formula :
x 100 d.
Statistical Analysis
Data were tested for normal distribution and homogeneity of variance. Two Way
Analysis of Variance (ANOVA) followed by Student-Newman Keuls multiple comparison were used to compare mean HSI between frog populations and mean between sites and seasons.
Quantitative histological data from cadmium contamination article and mean comparison between sex from herbicide contamination article were analyzed by Student’s t-test.
Correlation of melanomacrophage between MMC numbers and herbicide residues
(Thammachoti,2012) was determined using Pearson’s correlation
3.
Results and Discussion a.
HSI of Microhyla fissipes
The results from HSI of M.fissipes (Table 1) showed that mean of overall HSI in male frogs was significantly higher in the frog collected from high-Cd habitat (2.0769 ± 0.2958) than those from the low-Cd habitat (1.4462 ± 0.0772). Changes of HSI have been reported as a biomarker responded to various environmental contaminations. It may indicate the disturbances at cellular and tissue levels that lead to alterations in the organ gross structure.
Parameter
HSI
Period
Early wet season
Middle wet season
Late wet season
Low environmental
cadmium habitat
1.2965 ± 0.0937 (n=13)
High environmental
cadmium habitat
1.6145 ± 0.2049 (n=12)
1.5540 ± 0.0637 (n=16)
1.4881 ± 0.3183 (n=3)
1.9886 ± 0.4439 (n=4)
2.6276 ± 0.4951 (n=7)

Overall 1.4462 ± 0.0772 2.0769 ± 0.2958 *
Table 1. Hepatosomatic Index of Microhyla fissipes ‘s male
Based on table 2, the result of HSI of Microhyla fissipes ‘s female showed not significant different between low environmental cadmium and high environmental cadmium habitat.This result caused by the vary of body’s weight and liver’s weight . Body condition is a practical tool for biologists and managers to gauge the overall health of fish population, and a good indicator of fish habitat quality and pollution levels
Parameters Period Low environmental
cadmium habitat
High environmental
cadmium habitat
HSI Early wet season 1.8122 ± 0.2648 (n=11) 2.7026 ± 0.7962 (n=9)
Middle wet season 1.8786 ± 0.9535 (n=2) 1.6105 ± 0.2394 (n=3)
Late wet season 2.1300 ± 0.1659 (n=14) 2.7876 ± 0.4675 (n=6)
Overall 1.9403 ± 0.0968 2.3669 ± 0.3790
Table 2. Hepatosomatic Index of Microhyla fissipes ‘s female b.
Histological analysis of Microhyla fissipes’s liver

Fig.2. Micrograph of the liver tissues of Microhyla fissipes collected from low-Cd habitat. (A) Infiltration of white bloodcells (arrows) and melanomacrophage centers (arrow heads). Bar = 50 μm. (B) Lipid droplet accumulation (arrows) incytoplasm of hepatocytes. Bar = 50 μm. (C) Foci necrosis (asterisks) in some areas of the liver tissue. Bar = 100 μm. (D)Fibrosis in some areas of the liver tissue.
Bar = 50 μm. H&E
The histology result of M. fissipes ‘s liver collected from low environmental cadmium habitat showed some alterations including white blood cell infiltration, melanomacrophage centers (MMCs), lipid droplet accumulation in hepatocytes, fatty degeneration, foci necrosis and fibrosis of theliver tissues (Fig. 1).
Figure 2. Micrograph of the liver tissues of Microhyla fissipes collected from high-Cd habitat. (A) Infiltration of white bloodcells (arrows), melanomacrophage centers (arrow heads) and fatty degeneration (asterisks) in the liver tissue. Bar = 50 μm.(B) High degree of lipid droplet accumulation
(arrows) in the cytoplasm leading to fatty degeneration of hepatocytes. Bar=
50 μm. (C) Necrosis (asterisks) in perivascular areas of the liver. Bar = 50 μm.
(D) Fibrosis in perivascular areas of the liver. Bar = 50 μm
The histology result of M. fissipes ‘s liver collected from high environmental cadmium habitat showed the same alterations but in higher degree of severity for MMCs and lipid accumulation (Fig. 2). The higher degree of lipid droplet accumulation was found as a very large droplet accumulated in the cytoplasm resulting in fatty degeneration in some areas of liver parenchyma (Fig.2B). Leukocyte infiltration is a cascade of processes including leukocyte migration and adhesion to the micro vascular endothelial surface, matrix metallo proteinase production for BBB breakdown, leukocyte extra vacation into brain tissue, and finally the release of cytokines to brain tissue triggering an inflammatory response. The contribution of

leukocyte infiltration in reperfusion injury is also supported by the beneficial effects of neutrophil depletion, in which the animals after transient ischemia showed smaller infarct size when administered with either antineutrophil antiserum or monoclonal antibodies. Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation(Bataller,2005).
The major alteration observed in these frog populations is MMC formation.So, the result of qualitative histology showed that it is relatively higher in the frog lived in high-Cd habitat (see comparison in Figs. 1C and 2C). Necrosis, fatty degeneration and fibrosis, and
MMCs possibly involve with scavenging cell debris and cytotoxic substances due to chronic exposure to environmental cadmium. MMCs were also reported to accumulate some metals, they possibly accumulate cadmium (Loumbourdis and Vogiatzis 2002). Melanomacrophage in amphibian has many possible functions, for instance, lowering metabolic activities during hibernation, phagocytosing red blood cells leaked from blood vessel into liver, scavenging cytotoxic substances, cell debris, infection us agents and foreign substances (Loumbourdis and Vogiatzis, 2002). The results of hepatosomatic index indicate that HSI are not sufficient to interpret the effects of environmental contamination of a certain toxic compound. It needs to combine with using of histological biomarkers that clarified the toxic responses of animal organ system to the toxicant. Overall, the results indicate that contamination can interfere with the frog health. It can be important implication for overall health of animals/human in the areas of Mae Sot District, Tak Province,Thailand. c.
Histological analysis of Fejervarya limnocharis ‘s liver
The histological result showed significant higher of MMC in the liver tissue of
F.limnocharis from herbicide site than from reference site.Furthermore, there were significant seasonal differences in these numbers in both reference and contaminated site frogs (Table 3), which may relate to the difference in agricultural activities of both study sites
Figure 3. Micrographs of liver (H&E staining) of the rice frog, F. limnocharis , caught from the reference site (a = 100X, c = 400X) and the contaminated site (b = 100X, d =
400X) in Nan Province, Thailand. Melanomacrophages (arrow) and melanomacrophage centers (MMCs; arrow head) are shown.

Period
Late Wet a.
Number of Melanomacrophage of Fejervarya limnocharis
Month/ Year
July 2010
Reference site
(MMC/mm 2)
Contaminated Site
(MMC/mm 2)
78.27 ± 16.07 a (N=10) 141.26 ± 17.26 A,* (N=10)
Early Dry October 2010 15.82 ± 3.03
c (N=10) 62.25 ± 7.77 C,* (N=10)
Late Dry January 2011 62.23 ± 6.31 ab (N=10)
41.78 ± 6.78 bc (N=10)
55.27 ± 4.32 C (N=10)
106.97 ± 7.80 B,* (N=10) Early Wet April 2011
Table 3. Mean ± S.E.M. of the numbers of melanomacrophages and melanomacrophage centers
(MMCs) in liver of the rice frog F. limnocharis caught from the reference site and the contaminated site during 2010-2011 in Nan Province, Thailand.
Correlation analysis of melanomacrophage and MMCs numbers versus herbicide residues in frog tissue (Table 4; Thammachoti, 2012) showed significant correlation between numbers of melanomacrophages and MMCs vs. atrazine in the rice frog (Pearson’s correlation coefficient = 0.586, p< 0.05). But there was no significant correlation between numbers of melanomacrophages and MMCs vs. glyphosate (Pearson’s correlation coefficient = -0.479, p
> 0.05) and paraquat (Pearson’s correlation coefficient = -0.119, p > 0.05) residues. Some herbicides (atrazine, glyphosate and paraquat) were contaminated in frog tissues at both reference and contaminated sites. The higher level of herbicide indicate that an intensive herbicides usage could lead to contaminate in tissue of the frog living in the paddy fields
(Thammachoti, 2012). Although the low of atrazine can be endocrine disrupting chemicals that may interfere reproductive system of amphibians (Hayes et al., 2002).
Herbicide
Atrazine
Glyphosate
Reference Site
Min = 3.62 ± 0.50
Max = 7.26 ± 1.96
Min = 4.91 ± 1.17
Max = 8.26 ± 2.64
Herbicide Site
Min = 4.12 ± 1.61
Max = 14.10 ± 5.83
Min = 5.48 ± 2.74
Max = 10.90 ± 6.29

Paraquat Min = 47.88 ± 7.74
Max = 70.07 ± 15.30
Min = 66.92 ± 20.39
Max = 115.18 ± 40.20
Table 2. Range Maximum and Minimum of Mean ± S.E.M. of herbicide residues in tissues of the rice frog F. limnocharis caught from the reference site andthe contaminated site during 2010-
2011 in Nan Province, Thailand (Thammachoti, 2012).
There were significant seasonal and site-related differences in numbers of melanomacrophage and MMC in liver of the rice frogs that tend to affect frog’s immune system in agricultural areas. Liver of the frog is a very plastic organ in which both the epithelial and the histiocytic components.So,it’s very sensitive to certain annual biological rhythms(i.e. reproduction) for seasonal difference .It regulated by highly integrated mechanisms responsible for maintaining a functional homeostatic balance during the different adaptation responses (Barni et al., 2002). MMC’s function might be linked with cell and compound storage, destruction, and detoxification and iron recycling (Agius and Roberts, 2003). MMCs of teleost fish were reported to trap and retain antigens during the immune response and were closely associated with immunoglobulin-secreting cells (Vigliano et al., 2006).
Since MMCs could play roles inimmunity (inflammatory and humoral responses; Agius and Roberts, 2003), an increase in number and area of MMCs could be sensitive to environmental stressors. The occurrence of MMCs may vary depending on many factors namely the nutritional status, health or size of a particular fish species . The poor health fish or fish with nutritional deficiencies and larger fish tend to have more or larger MMCs.
Moreover, the number and/or size of MMCs in many fish increase with age (Brown and
George, 1985; Blazer et al., 1987).Importantly, numerous studies had documented an increase in their number, size or hemosiderin content infish collected at contaminated sites compared to those collected at reference sites. MMCs were used as a potentially sensitive biomarkers of contaminant exposure and a potential immunotoxic biomarkers (Matavulj et
al., 2005).
In further study, MMCs can be used as biomarkers of vertebrate health and environmental degradation should be further examined using the systematic controlled experiment (Blazer and Dethloff, 2000). Herbicide contamination on immune system of the rice frog should be performed with other techniques to examine both non-specific and specific immune response of the rice frog.
Conclusion
The mean number of MMCs was not statistically significant different between two population but qualitative histology of Microhyla fissipes ‘s MMCs is relatively higher in the frog lived in high-Cd habitat. The contamination effect on melanomacrophage and MMC of
Fejervarya limnocharis is higher in herbicide site than reference site at Nan Province

Acknowledgement
I would like to express my deepest appreciation to all Ajarn those who provided me the possibility to complete this seminar project. A special gratitude I give to my supervisors
Assist.Prof.Dr.Noppadon Kitana and Dr.Jirarach Kitana, whose contribution in stimulating suggestions and encouragement, helped me to prepare my seminar project.Furthermore I would also like to acknowledge with much appreciation for Biosentinel laboratory team who support me and give suggestion
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