Journal of Life Sciences Volume 10 Numbe
JLS
Journal of Life Sciences
Volume 10, Number 2, February 2016 (Serial Number 93)
Contents
Zoology
59 The Length-weight Relationship of Diplodus vulgaris (Teleostei, Sparidae) from Benghazi Coast (Libya)
Abdalla Nassir Elawad, Anwaar M. Saeid and Ramadan A. S. Ali
66 Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison between in utero and Neonatal Exposures
Cristiano Foschi, Manuel Luís Orta, Licia Radicchi, Germana Szpunar and Mauro Cristaldi
Biotechnology
77 Stochastic Lattice gas Cellular Automata Model for Epidemics
Ariel Félix Gualtieri and Juan Pedro Hecht
85 Neoadjuvant Chemotherapy for Transitional Cell Carcinoma of the Bladder: A Single Centre Experience
Gauhar Sultan, Babar Malik, Syed Najeeb Niamatullah, Altaf Hashmi, Asad Shehzad, Mubarak M and Syed Adeeb ul Hassan Rizvi
Interdisciplinary Researches
91 Identification of 11 STD Pathogens in Semen Using Polymerase Chain Reaction (PCR) and “Flow-through” Hybridization Technology
Rubina Ghani, Kashif Nisar, Hasan Ali and Saara Ahmad 100
The Effect of Family Planning Methods on Food Security in Oyo State, Nigeria
Adepoju Adebusola Adenike
Journal of Life Sciences 10 (2016) 59-65
doi: 10.17265/1934-7391/2016.02.001
DAVID PUBLISHING
The Length-weight Relationship of Diplodus vulgaris (Teleostei, Sparidae) from Benghazi Coast (Libya)
Abdalla Nassir Elawad, Anwaar M. Saeid and Ramadan A. S. Ali Omar Al-Mukhtar University, Faculty of Science-Department of Zoology, Marine Biology Branch
Abstract: Length-weight relationships (LWRs) were determined for fish species Diplodus vulgaris from the Benghazi coast along the eastern Mediterranean Sea coast of Libya. Samples were collected using trammel nets. The parameters a and b from the LWR formula W = aLb were estimated. The values of the exponent b of the length-weight relationships in all categories range between 2.295 in September to 3.208 in August. The total number of fish samples investigated were 290, from which 179 were males, 41 females and 70 fishes immature. The sex ratio male to female were 4.3:1. In January, August and Females the slope “b” close to equal 3, were the categories exhibited isometric relationship, in September, October and November were slope “b” not equal 3, which the categories exhibited negative allometric relationship. The mean observed length of male was 18.29 cm at mean observed weight 114.8 g, and mean observed length of female was 19.15 cm at mean observed weight 133.97 g.The general equation for the
length-weight relationship for both sex was: W = 0.03835L 2.77 , for male was: W = 0.036 L 2.74 , for female, W = 0.016 L 3.02 .
Key words: Diplodus vulgaris, allometric, ismeteric, Benghazi coast, sex ratio.
1. Introduction eastern Atlantic coast from France to Senegal, including the Madeira, the Azores and the Canaries
The relation between the length of fish and the Archipelagos. It is also present from Angola to South weight used since before year 1930, first was Africa [6]. It can be found close to rocky and sandy described by the cubic parabola [1]. But after that bottoms to a maximum depth of 60 m. Juveniles often another equation was used instead of cubic parabola live in coastal lagoons and estuaries [7] and it is called general parabola, it gives better results [2]. The considered a resident species in artificial reefs [8]. values of a and b differ between species, through the Mainly caught by line and hooks, generally recognize year and through the spawning season [3]. The as commercial value, frequently in huge catch relation between length (L) and weight (W) of fish is inhabiting the eastern coast of Libya [9]. very important for estimating growth rates, age Although there were many studies dealt with structures, and stock conditions; comparing life different aspects of Libyan fisheries for family histories of fish species between regions; and sparidae and for the species D. vulgaris, almost assessing the condition of fish and other components concentrated on general biology, as food and feeding of fish population dynamics [4]. habits, reproduction, length weigh. But no study With the knowledge that, in the Mediterranean sea concentrated on studied the length weight as such there are 25 species of family sparidae, of which 14
during all months of year.
species inhabiting the Libyan coast, such as The main aims of the present study are to determine Diplodusvulgaris [5]. The common two-banded the relationship between length and weight during sex seabream, D. vulgaris is a demersal species distributed
and months.
in the Mediterranean and Black Seas and along the
2. Material and Methods
Corresponding author: Abdalla Nassir Elawad, Ph.D., research field: population dynamic fishes.
The study area is located on the east coast of the
60 The length-weight relationship of Diplodus vulgaris (Teleostei, Sparidae) from Benghazi Coast (Libya)
Libyan line, include all coast of Benghazi and areas there, all these reasons made the availability in around which located between 32°36 ′ N and 20°03′ E
collecting fishes sample, than other areas. Trammel on the Mediterranean Sea (Fig. 1). The coast line
and gill nets are still working in the area till now. slope is characterized by strongly phenomena lagoon
The relationships between body weight and total marshes and sand dunes (Guda, 1973). Just line a
length for the species D. vulgaris were established depth of 20 m from the coast line in front of the city of
following Gulland [2], for the whole total sample, Benghazi more than 5 Ikm, however. This area
monthly and by sexes (male and females). The includes seven fishing associations and many fishing
constants “a” and “b” were obtained from the companies. In the eastern part of Libya (Benghazi
equation:
region) a list of bony fishes came up with a total of b W(i) = q*L(i) 201 species belonging to seventy one families and
Where W(i) is the body weight, L(i) is the total fifteen orders [10].
length “a” and “b” are constants. The exponential The data and information was gathered from the
equation was converted into a linear by logarithmic areas around and near Benghazi coast, 32°36 ′ N and
transformation.
20°03 ′ E on the Mediterranean sea (Fig. 1), because it Ln W(i) = ln q+ b*ln L(i) has been consider the largest areas in the east coast,
or
The areas is packed with a large number of fisherman, Y(i) = a + b*X(i) reach 1,200. Also all kinds of fishing were practice
Where Y(i) = ln W(i), x(i) = ln L(i) and a = ln q.
Fig. 1 The study area in Benghazi coast.
The length-weight relationship of Diplodus vulgaris (Teleostei, Sparidae) from Benghazi Coast (Libya)
3. Statistical Analysis
relationships were found significant (P < 0.001) in the all groups. The slope “b” in all categories range between
The Effect of Sex and Months: General linear model was performed to determine
2.295 in September to 3.208 in August. In January, August and Females the slope “b” close to equal 3,
the effects of the sex of fish and the month of captured were the categories exhibited isometric relationship, in
using SPSS computer software (2012), release 20. Duncan Multiple Range Test was used to estimate the
September, October and Novemberwere slope “b” not equal 3, which the categories exhibited negative
differences between means. allometric relationship and for the other months, males,
and both sex showed positive allometric relationship From table 1 and 2, it is appeared that, the total
4. Results
were seen. However, allometry. was very small with number of fish samples investigated were 290, from
coefficients close to 3 and there were no statistically which 179 were males, 41 females and 70 fishes
significant differences in slopes or intercepts between immature. The sex ratio male to female were 4.3:1.
males and females. The analysis of variance (Table 2) The mean observed length of male was 18.29 cm at
indicated a high significant effects of months on both mean observed weight 114.8 g, and mean observed
total length and total weight, while the sex had no length of female was 19.15 cm at, mean observed
significant effects on the bellow mentioned traits. The weight 133.97 g. The correlation coefficient “r”,
general equation for the weight-length relationship for which measured the association between Length-weight 2.77 both sexes was: W = 0.03835L
regression parameters was estimated for all months, For male was: W = 0.036 L 2.74 and For female: W = males, females and the whole sample are presented in
0.016 L 3.02 .
Table 1 and Figures 2, 3 and 4, r seems to be high Within columns, means had different superscripts correlation in all categories r 0.5. The length-weight
differed significantly (P < 0.05).
Table 1 The relationship between Weight (Y) and Length (X) monthly, by sexes and for whole for Diplodus vulgaris from
Benghazi coast 2014-2015. Month
Significance December
Equation
P < 0.001 January
Y = 2.966X - 1.708
P < 0.001 February
Y = 3.112x - 1.881
P < 0.001 March
Y = 2.646x - 1.313
P < 0.001 April
Y = 2.573x - 1.243
P < 0.001 May
y = 2.983x - 1.752
P < 0.001 June
y = 2.623x - 1.246
P < 0.001 July
y = 2.8811x - 1.5782
P < 0.001 August
y = 2.9486x - 1.7083 R = 0.9219
P < 0.001 September
y = 3.208x - 2.071
y = 2.2956x - 0.8531
P < 0.001 October
P < 0.001 November
y = 2.3529x - 0.9896 R² = 0.9128
P < 0.001 male
y = 2.489x - 1.1738 R² = 0.9422
P < 0.001 female
y = 2.741x - 1.442
P < 0.001 whole
y = 3.016x - 1.791
y = 2.722x - 1.416
P < 0.001
62 The length-weight relationship of Diplodus vulgaris (Teleostei, Sparidae) from Benghazi Coast (Libya)
Table 2 Mean observed weight and length and calculated weight and length for D. vulgaris from Benghazi coast 2014-2015.
Categories
Mean observed weight
Mean observed length
Mean calculated weight Mean calculated length
(cm) Effect of month
(g+-S.D.)
(cm+-S.D)
71.89 ± 24.9 a 15.71 ± 1.8 a 78.917
18.03 February
115.55 ± 58.8 bc 17.97 ± 2.9 b 114.5
20.07 March
155.45 ± 45.05 de 20.89 ± 2.3 d 173.77
18.03 April
111.55 ± 115.8 bcd 17.86 ± 4.8 bc 109.46
17.3 May
103.23 ± 7.7 bc 17.67 ± 3.3 b 109.29
101.81 ± 61.8 bcd 16.67 ± 3.7 b 93.00 17.223 June
50.1 ± 12.7 a 13.63 ± 1.1 a 53.24 13.33 July
19.73 August
148.46 ± 54.8 cde 20.38 ± 2.5 cd 162.27
21.68 September
192.6 ± 53.5 ef 22.56 ± 2.08 de 215.03
22.26 October
207.25 ± 42.02 f 23.90 ± 2.1 e 252.30
77.4 ± 18.5 a 15.76 ± 1.6 a 79.61 14.94
16.77 Effect of sex Male
November 94.62 ab 18.26 ± 1.9 bc 119.70
17.94 Female
114.8 ± 66.96 a 18.29 ± 3.4 a 120.25
19.017 Over all mean
133.97 ± 78.10 a 19.15 ± 3.8 a 136.57
W = 0.038L2.722
Total weight
Total length (cm
Fig. 2 The relationship between total weight (gm) and total length (cm) for both sexes for D. vulgaris from Benghazi coast 2014-2015.
The length-weight relationship of Diplodus vulgaris (Teleostei, Sparidae) from Benghazi Coast (Libya)
W = 0.036L2.741
Total Weight
0 5 10 15 Total length (cm) 20 25 30 35
Fig. 3 The relationship between total weight (gm) and total length (cm) of male for D. vulgaris from Benghazi coast 2014-2015.
W= 0.016L3.016
Total Weight (g
Total length (cm) ر
Fig. 4 Weight-length relationship for female for D. vulgaris from Benghazi coast 2014-2015.
64 The length-weight relationship of Diplodus vulgaris (Teleostei, Sparidae) from Benghazi Coast (Libya)
5. Discussions
(2.8), male (2.7) and female (3.0) which indicated slight positive allometry with both sex and male,
The percentage of sex ratio male to female in this a while for female which indicate isometric growth. study was 4.3:1, with favor of male. Our results on This value was lower for values of (both sex and male) this study compare with the results of Taieb, et al. and similar with some values than the values of “b” of 2013, from Tunisia sea, he, recognized that the sex
D. vulgaris which is estimated by Mahmoud [13] in ratio for the species D. vulgaris was 1:1.66, and with Abu Qir Bay Egypt, (2.9), in Gulf of Tunis (3.05 both Dulcic, et al. 2011, from the Eastern Adriatic Sea, sex, 3.058 for male and 3.078 for female) [14], in the they mentioned 1.22:1, with favor to male, these Gulfof Lion (3.123) [15] and in the Egyptian variation in results me be due to different in locations Mediterranean water (3.003) [16]. The differences in b or types of gears used in captures of this species in its value observed for the species across areas may be areas ranges. Sadovy and Shapiro, they mentioned that attributed to the different atrophic conditions [17]. In the percentage of males to females varied with size of fact, the Mediterranean Sea, and the Eastern Basin in fish and also by season and months [11]. particular, is considered as one of the most oligotrophic The length-weight relationships a practical index of regions in the world in terms of both primary the condition of fish. In fisheries studies; the condition productivity and chlorophyll a concentrations [18]. factor is an essential biological parameter needed to
understand the suitability of the environment for good
References
living of fish [12]. In the present study the [1] Richer, W. E. 1975. Interpretation of Biological Statistics Length-weight regression parameters estimated for all
of Fish Populations, Department of the Environment months, male, female and both sex were showing
Fisheries and Marine Service Pacific Biological Station, different result in slope “b” and showed high
Nanaimo, B. C. V 9R5 K6, pp. 266.
correlation coefficient “r” between total weight and [2] Gulland, J. A. 1985. Fish Stock Assessment. A Manual of Basic Methods. Marine Resources Service. Rome, Italy, p.
total length of species D. vulgaris, presented in table1
and Figs 2, 3 and 4. The length-weight relationships [3] Ahemed, A. H. 1987. Fish Biology, University of El were found significant (P < 0.001) in the all groups. In
Basra: pp. 279.
January, August and Females, were exhibited [4] Bagenal, T. B., and Tesch, F. W. 1978. Age and Growth. In: Methods for Assessment of Fish Production in Fresh
isometric relationship, in September, October and Waters, third edit. (Bagenal, T. ed.), pp. 101-36. IBP November were exhibited negative allometric
Handbook No. 3. Oxford: Blackwell Scientific relationship while for the other months, males, and
Publications.
both sex showed positive allometric relationship. [5] Ibrahim, S. M. 2013. Study, characterization and biolical study on some species of family Sparidae in Ain
However, allometry was very small with coefficients El-Ghazala Gulf of eastern Libya. M.Sc. thesis. close to 3 and there were no statistically significant
[6] Bauchot, M. L., and Hureau, J. C. 1986. Sparidae. In: differences in slopes or intercepts between males and
Whitehead, P. J. P., Bauchot, M. L., Hureau, J. C., females. This finding recognized by many authors [4] Nielsen, J., and Tortonese, E. (Editors). Fishes of the North-eastern Atlantic and the Mediterranean. UNESCO,
were suggested that the pattern of weight relative
Paris, 2: 883-907.
growth may fluctuate over the year according to [7] Monteiro, P. 1989. La fauneichthyologique de la factors such as food availability, feeding rate, gonad
laguneRia Formosa (Sud Portugal). development or spawning period. Repartitionetorganizationspatio-temporelle des communautés: application à l’aménagement des resources.
In the present study, the value of the exponent “b” Thèse Doctorat. Université des Sciences et Techniques du for both sex for species D. vulgaris was found to be
Languedoc, Montpellier, 219 p.
65
The length-weight relationship of Diplodus vulgaris (Teleostei, Sparidae) from Benghazi Coast (Libya)
[8] Santos, M. N. 1997. Icthyofauna of the artificial reefs of and Growth of Diplodus vulgaris (Sparidae) in the Gulf the Algarve coast. Exploitation strategies and
of Tunis.” Cybium 34 (1): 37-45. management of local fisheries. Ph.D Thesis. Universidade
[15] Man Wai, R., and Quignard, J. P. 1982. “The Seabream do Algarve, UCTRA, Faro, 223 p.
Diplodus sargus (Linne 1758) in Gulf of Lion: Growth of [9] Shakman, E., and Kinzelbach, R. 2007. “Commercial
the Seabream and Characteristics of Landings from the Fishery and Fish Species Composition in Coastal Waters
Commercial Fishing Grounds of Sete and Grau-du-Roi.Rev.” of Libya.” Rostocker Meeresbiologische Beiträge 18:
Trav. Inst. Peches Marit. Nates 46 (3): 173-19. 63-78.
[16] El-Maghraby, A. M., and Botros, G. A. 1981. [10] Al-hassan, L. A., and El-silini, O. A. 1999. “Check-list of
“Maturation, Spawning and Fecundity of Two Sparid Bony Fishes Collected from the Mediterranean Coast of
Fish, D. sargus, D. vulgaris and Geoffer Bull.” Nat. Inst. Benghazi, Libya.” Revista de Biologia Marina y
of Oceanography and Fisheries, ARE 8 (2): 51-67. Oceanografia 34: 291-301.
[17] Chilari, A., Petrakis, G., and Evaggelos, T. 2006. [11] Sadovy, Y., and Shapiro, D. Y. 1987. “Criteria for the
“Aspects of the Biology of Blackspot Seabream (Pagellus Diagnosis of Hermaphroditism in Fishes.” Copeia 1987:
bogaraveo) in the Ionian Sea, Greece.” Fish. Res. 77: 136-56.
84-91.
[12] Le Cren, E. D. 1951. “The Length Weight Relationship [18] Azovy, Y. 1991. “Eastern Mediterranean: a Marine and Seasonal Cycle in gonad weight and Condition in the
Desert?” Mar. Pollut. Bull. 23: 225-32. Perch (Perca fluviaittlis).” J. Anim. Ecol. 20: 201-19.
[19] Dul čić, J., Pallaoro, A., Matić-Skoko, S., Dragičević, B., [13] Mahmoud, H. H., Osman, A. M., Ezzat, A. A., and Salah,
Tutman, P., Grgi čević, R., Stagličić, N., Bukvić, V., A. M. 2010. “Biological Status and Management of
Pavli čević, J., Glamuzina, B., and Kraljević, M. 2011. Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817) in Abu
“Age, Growth and Mortality of Common Two-banded Qir Bay, Egypt.” Egyptian, Jou. of Aquatic Res. 36 (1):
Seabream, Diplodus vulgaris (Geoffroy Saint-Hilaire, 115-22.
1817), in the Eastern Adriatic Sea (Croatian coast).” [14] Mouine, N., Mohamed, H., and Nadia, M. C. 2010. “Age
Journal of Applied Ichthyology 27: 1254-8.
Journal of Life Sciences 10 (2016) 66-76
doi: 10.17265/1934-7391/2016.02.002
DAVID PUBLISHING
Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison between in utero and Neonatal Exposures
1 2 3 1 Cristiano Foschi 1 , Manuel Luís Orta , Licia Radicchi , Germana Szpunar and Mauro Cristaldi
1. Department of Biology and Biotechnology Charles Darwin, Faculty of Mathematical, Physical and Natural Sciences, University of Rome, Rome 00161, Italy 2. Department of Cell Biology, Faculty of Biology, University of Seville, Seville 41012, Spain 3. Civic Museum of Zoology, via U. Aldrovandi 18, Rome 00197, Italy
Abstract: The purpose of this study was to develop a biological model to evaluate the genotoxic effects of natural emissions of Radon-222 and its decay products. To this aim, mice of the Swiss CD1 strain were exposed to Radon for different periods (adult life, early postnatal and in utero exposure) and two simple but accurate mutagenicity tests (Micronucleus test and the Comet assay) were applied to the peripheral blood of mice. The study was carried out in two small towns in Latium region—Italy, where radon pollution is notoriously present due to the volcanic soils. One experiment was carried out in the cellar of a house in Ciampino (Rome) and two experiments were performed in an old cellar in Vetralla (Viterbo). The results showed that in all mice groups exposed to natural emissions of radon and its decay products, the micronucleated erythrocytes frequency (ME) was significantly higher than that observed in the mice control. The single cell gel electrophoresis (Comet assay) was performed in lymphocytes of adult mice in the last experiment. The results for this test also show that DNA damage was higher than in the cells of the mice control and the cells of mice exposed for a shorter period of time. To confirm these findings, the single cell gel electrophoresis (Comet assay) was performed in lymphocytes of adult mice in the last experiment. Similarly, this result could be linked to a greater sensitivity of neonatal mice to radon emissions compared with intrauterine mice. Further investigations on the effects of radon and its decay products during the intrauterine life and the first neonatal period should be performed to better clarify its genotoxic activity.
Key words: Radon, genotoxicity, DNA damage, micronucleus test, comet assay.
1. Introduction or Thorium. Radon rises up from the soil surface (some building materials emit large quantities) and
Radon is a radioactive noble gas, chemically inert, tends to accumulate in confined spaces. In addition it decay product of Uranium-238. It is classified by can enter buildings through the called “chimney effect” WHO/IARC as group 1 carcinogen. Home (indoor) due to pressure imbalance that is created for the exposure to short lived radioactive disintegration highest temperature of the air in confined spaces. This products of Radon-222 is responsible for about half of problem affects mainly basements and floor plans, and all non-medical exposure to ionizing radiation [1]. therefore people who work in tunnels, underpasses Uranium-238 is present throughout the earth’s crust, and caves (catacombs guides, bank employees, etc.) but environmental exposure to Radon-222, and its are continuously exposed to these radioactive isotopes. decay products, is especially stronger in dwellings Radon has a half life of 3.8 days, allowing it to located in areas where the subsoil is rich in Uranium diffuse from the soil into the air before decaying by
emission of α particle into a series of its short lived Corresponding author: Cristiano Foschi, Ph..D., research fields: radon, genotoxicity, natural radioactivity, DNA damage
radioactive progeny. Two of these, Po-218 and Po-214, and mutagenicity testing.
also decay by emitting
α particles.
Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison
between in utero and Neonatal Exposures
Most of the inhaled Radon is immediately exhaled, All cages contained water and food ad libitum. The however it tends to be deposited on the bronchial
cages were placed in a cellar, six meters below road level. epithelium, thus exposing cells to irradiation [2].
The three pairs gave birth to two, 15 and 11 mice, Since the beginning of the last century a relation
respectively. In total 28 newborn mice were exposed between exposure to high radon concentration and
to radon for 23 days (18 to 19 days of intrauterine lung cancer was observed [3, 4]. Studies of exposed
exposure and four to five days of exposure after birth). miners have consistently confirmed associations
2nd experiment (18-12-2009/20-01-2010): between radon and lung cancer [5, 6]. However,
Eight adult females CD-1 Swiss, were placed in two results obtained on the biological effects are hard to
cages (four mice per cage), with water and food ad explain, because of the differences in several libitum. The cages were placed in a cellar, six meters parameters, such as duration and periodicity of
below the road level, and exposed to radon for about exposures, concentration values of radon and products
33 days. The experiments were performed six meters decay, synergic effects of tobacco smoke, etc.
below ground level in Vetralla, and about two meters Genotoxic properties of Radon have been investigated
below ground level in Ciampino.
because they are important in order to understand the events that may involve radon to the onset of
2.2 Determination of Radon-222 Activity neoplasia in cytogenetic studies [7-11].
Ciampino cellar: Radon-222 activity measurements In this study we present data that demonstrate that
were carried out for over 22 days in the proximity of Radon exposures in two Italian areas cause the cages, by an active detector (Radon Scout made by
genotoxicity in vivo as measured with two standard SARAD GmbH, Dresden, Germany). techniques. Furthermore, our data also suggest that
Vetralla cellar: Radon-222 activity was measured animals in the neonatal period are more sensitive to
for by an active detector (AWARE Corporation Radon as compared to fetuses [12, 13].
RADIM 5 made by Plch Smm, Czech Republic) and
2. Materials and Methods
CR 39 passive detectors provided and analyzed by U-Series, Bologna, Italy.
2.1 Study Sites: Experiments were Carried out in In both instruments (active detectors) the
Ciampino (Rome) and Vetralla (Viterbo) (Fig. 1) concentration of radon is determined by measuring the
2.1.1 Ciampino Experimental Design α-activity of the decay products of the conversion of (13-09-2007/05-10-2007)
radon, Polonium-218 and Polonium-214, collected Two females CD-1 Swiss in advanced pregnancy
from the detection chamber on the surface of a (around the 15th gestation day), caged with water and
semiconductor detector by an electric field. The only food ad libitum were placed in the cellar of a building.
difference in these two detectors is the time interval Within three days of the start of the experiment, both
used between measurements.
females gave birth to 13 newborns. In total 26 Radon concentrations were calculated by the newborn mice were exposed to radon gas for 21 days
laboratory “U-Series” (Bologna, Italy). (three days of intrauterine exposure and 18 days of
2.3 Genotoxicity Testing
exposure after birth).
2.1.2 Vetralla Experimental design Micronucleus test and the Comet assay were applied 1st experiment (25-12-2007/29-01-2008):
to the peripheral blood of mice. Where possible (for Three pairs of adult CD-1 Swiss, were placed in
adults) the exposed group was tested before being three cages. A fourth cage contained the four adult males.
placed in cages to determine average levels of DNA
68 Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison
between in utero and Neonatal Exposures
Fig. 1 Map of Latium region; Ciampino is indicated by outlined arrow, Vetralla by solid arrow (source: www.tuttocitta.it).
damage prior to exposure. These mice were analyst at 1,000x magnification and micronuclei immediately tested again after the exposure period.
frequencies were determined counting 2,000 Control mice (newborns and foetuses) of the same
erythrocytes per animal.
strain and age were held in stabulary and tested in the In total, 72 mice were exposed and analyzed by same manner at the end of the exposure time only.
micronucleus test (excluding control mice). For adults, Micronucleus test: smears were prepared by blood
blood samples collected from each mouse before the samples collected by caudal vein puncture of each
exposure were used as controls. For newborns and animal, avoiding any useless suffering and pain.
foetuses, N = 9 mice of the same strain were held in Slides were coded, fixed in absolute methanol and
controlled stabulary conditions and used as control stained with the May-Grunwald-Giemsa stain. mice for the Ciampino experiment. N = 18 were used Micronuclei (MN) were scored blindly by the same
as control mice for Vetralla.
Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison
between in utero and Neonatal Exposures
Comet assay: blood samples were collected by migrated DNA). Five different classes of DNA caudal vein puncture, as before described, and placed
damage were established following classification in Eppendorf tubes (1 mL). Heparin was used as an
given by Anderson et al. [16]. Class 1 corresponded to anticoagulant and DMSO (10%) was added before
cells showing DNA damage between 0 and 5%, class freezing.
2 (5-20%); class 3 were cells with DNA damage Slide preparation: regular slides were coated with a
between (2-40%); class 4 (40-95%); and class 5 1% solution of standard agarose in distilled water, by
(95-100%), which corresponds to cells with the immersing vertically for 2 s and air-drying to solidify
highest damage or totally damaged cells. For each the agarose. Once the slides were dry and totally
experimental point at least 324 Comets were transparent, they were kept at 4 °C and used up to 1
measured.
month after preparation. We employed a modification
2.4 Statistical Analysis
[14] of the protocols described by Singh et al. [15] and Fairbairn et al. [16]. Only 40-100 μL of cell solution
Micronucleus test: since some of the variables were was embedded in a 0.7% low melting agarose solution
not normally distributed (Lilliefors test, P < 0.05), we in PBS and immediately pipetted onto the coated
used nonparametric tests. Medians were analysed by slides and then a coverslip was placed on. The slides
Mann-Whitney U-test.
were incubated at 4 °C for 10 min. Coverslips were Comet assay: Mann-Whitney test and χ (chi-square
removed and a third (low melting) agarose layer was test) have been used for statistical evaluation. added, together with new coverslips. Slides were
All statistical analyses were carried out using again incubated at 4 °C for 10 min. Coverslips were
STATISTICA 6.0 package (StatSoft, Tulsa, OK, finally removed and cells were immersed in lysis
USA).
solution (10 mM Tris-HCl, 2.5 M NaCl, 100 mM
3. Results and Discussion
Na 2 -EDTA, 0.25 M NaOH, 1% (v/v) Triton X-100
3.1 Exposures
and 10% (v/v) DMSO, pH 12.0) for 1 h at 4 °C in the dark.
Ciampino site: Figure 2 shows the 222 Rn Electrophoresis: in order to unwind the DNA, the
concentrations measured using electronic detector slides were incubated for 20 min in electrophoretic
Radon Scout in continuum. The mean concentration of
the entire period of the exposition of the newborns NaOH, pH 12.8 (Alkaline conditions). Electrophoresis 3 was 562 Bq/m . The graphic shows large yet short
buffer containing 1 mM Na 2 -EDTA and 300 mM
was carried out at 1 V/cm for 20 min. Slides were then variations of radon activity, ranging from the neutralized with 3 × 5 min washes of 0.4 M Tris-HCl 3 maximum value of 5,054 Bq/m and the minimum
pH 7.5 to remove alkali and detergent. After that, cells 3 value (0 Bq/m ). Total exposure at the end of period
were stained with the fluorochrome 4' was 260 kBq h/m 3 .
6-diamidino-2-phenylindole (DAPI) in Vectashield 222 Vetralla site: 1st experiment: Figure 3 shows Rn (mounting medium for fluorescence H-1000, Vector
concentrations measured using electronic detector Laboratories). Slide scoring: DNA damage was
RADIM 5 in continuum. The graphic shows (large analysed using the Comet Score program (an online
short) variations of radon activity, ranging from the free Comet scoring software web). Two parameters
maximum value of 12,972 Bq/m 3 and the minimum were estimated for each Comet: (1) DNA damage 3 value of 31 Bq/m . The mean value of the total period
(percentage migrated DNA in the tail), and (2) tail 3 of the exposure of the newborns was 2,074 Bq/m . moment (tail length × tail intensity or percent
Total exposure since the start to the end of gestational
70 Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison between in utero and Neonatal Exposures
Fig. 2 Graph showing the trend of the concentration of 222 Rn (Bq/m ) monitored by the active detector Radon Scout in Ciampino from 12/09/2007 to 07/10/2007.
Fig. 3 Graph showing the trend of the concentration of 222 Rn (Bq/m 3 ) monitored by the active detector RADIM 5 in Vetralla from 25/12/2007 to 30/01/2008.
Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison
between in utero and Neonatal Exposures
the exposed group compared with the control group (P mice (entire period of 35 days) was 1,700 kBq h/m 3 .
period was 1,090 kBq h/m 3 . Total exposure for adult
2nd experiment: 222 Rn concentrations were Vetralla site: Table 2 shows the mean frequencies measured using just passive detectors (CR-39). The
of peripheral blood micronucleus test in all mice exposure was calculated every 10 days (Table 1). The
offspring born in Vetralla cellar and in the control mean concentrations are included between 745 Bq/m 3 offspring. It was observed that the frequency of
to 2,100 Bq/m 3 . Total exposure after the last ten days micronucleated erythrocytes is significantly higher in was 200 kBq h/m 3 , after twenty days 560 kBq h/m 3 the exposed group compared with the control group (P
and, since the start to the end of the period, was 1,160
kBq h/m 3 . Vetralla 1st experiment: Table 3 shows the mean With regards to the experiments of newborns and
frequencies of peripheral blood micronucleus test foetuses (Vetralla) the entire exposure period (about
before exposure and after 35 days in the Vetralla cellar.
25 days) consisted of about 19 days of intrauterine It was observed that the frequency of micronucleated exposure and five to six days of exposure after birth.
erythrocytes is significantly higher in the exposed These data were compared with those obtained in
group compared with the control group (P < 0.05). newborn mice from the cellar in Ciampino exposed to
Vetralla 2nd experiment: Table 4 shows the mean 222 Rn for three to four days in utero and 18 days of
frequencies of peripheral blood micronucleus test postnatal exposure (about 22 days in total). The mice
before exposure and after 33 days in the Vetralla located in the Vetralla cellar were exposed to a 222 Rn
cellar. It was observed that the frequency of
micronucleated erythrocytes is significantly higher in higher compared with the mice exposed in Ciampino’s
concentration (1,090 kBq h/m 3 ) almost four times
the exposed group compared with the control group (P cellar (260 kBq h/m 3 ).
In the experiment carried out in Ciampino, the
3.2 Micronucleus Test genotoxic damage observed in newborn mice exposed
Micronucleus test was carried out on newborn mice to a low indoor radon concentration (300 kBq h/m 3 ) (N = 26 from Ciampino and N = 28 from Vetralla) and
may be related to the period in which the animals were adult mice (N = 18) from two experiments carried out
exposed. It should be noted that in this case the in Vetralla.
females were exposed on the 15th the day of pregnancy. Ciampino site: Table 1 shows the results obtained
Therefore the newborn mice were exposed for fewer applying peripheral blood micronucleus test in the
days during the intrauterine life (4-5) and for 18 days newborn mice exposed in Ciampino cellar and in the
after birth, compared with the mice in the Vetralla control mice. It was observed that the frequency of
cellar which were exposed for the whole intrauterine micronucleated erythrocytes is significantly higher in
phase and only for few days (4-5) after birth.
Table 1 222 Rn concentrations measured by passive detectors (CR-39) and relative exposures.
Vetralla 2 nd experiment
[kBq h/m 3 ] Total exposure [kBq h/m 3 ] 18/12/2008-29/12/2008
Time of exposure
Concentrations
Exposure
222 Rn [Bq/m 3 ]
29/12/2008-09/01/2009
09/01/2009-19/01/2009
72 Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison
between in utero and Neonatal Exposures
Table 2 Mean frequencies ± SD of micronucleated erythrocytes in newborn mice exposed in Ciampino cellar. N = number of animals; ME = Micronucleated Erythrocytes; E = Erythocytes; Control = mice not exposed (from stabulary at the Sapienza University, Rome); significance respect to control: b = P < 0.01; d = P < 0.0001.
Ciampino
N ME/1,000 E Exposure [kBq h/m 3 ] Control
N ME/1,000 E Exposure [kBq h/m 3 ] Control
Table 3 Mean frequencies ± SD of micronucleated erythrocytes in adult mice exposed in Vetralla cellar. N = number of mices; ME = Micronucleated Erythrocytes; E = Erythocytes; Control = blood samples before exposure; significance respect to control: a = P < 0.05.
Vetralla 1st experiment
N Male/female ME/1,000 E Exposure [kBq h/m 3 ] Control (before exposure) 10
Table 4 Mean frequencies ± SD of micronucleated erythrocytes in adult mice exposed in Vetralla cellar. N = number of mice (females); ME = Micronucleated Erythrocytes; E = Erythocytes; Control = blood samples before exposure; significance respect to control: c = P < 0.001.
Vetralla 2nd experiment
N ME/1,000 E Exposure [kBq h/m 3 ] Control (before exposure)
3.3 Comet Assay control mice and exposed mice, belonging to the various classes of damage (absent, low, medium, high
In the present study we have made use of the rapid
and total).
method applicable to mammalian lymphocytes, which Data obtained from the experiments in both the do not need to be isolated with the potentially
Ciampino and Vetralla cellars show that perinatal cytotoxic drug Ficoll, as previously described by Daza
exposure to radon emissions induces a significant et al. [13]. As we can see in Table 5, DNA damage
increase in micronuclei frequencies (ME/1,000E) both assessed by the Comet assay in mice after final
in newborn mice and in mice exposed during
exposure (1,160 kBq h/m 3 ) was statistically intrauterine phase.
significantly higher (Mann-Whitney test) than that Some differences in the two experiments should be observed in control samples (taken before exposure).
noted: in Ciampino, the exposure was almost totally Significant statistical differences were observed neonatal (from birth until 18 days after) compared
considering both the parameters scored (percent of with the exposure in Vetralla which was mainly damage DNA in the Comet tail, and tail moment of
intrauterine (about 19 days).
the Comets). Despite these differences both experiments gave the
Another statistical analysis ( 2 χ or chi-square test)
same result; in mice from Ciampino, a ME frequency has been carried out considering the five classes of
increase was observed with a radon exposure lower DNA damage reported by Anderson et al. [17]. Table 3 (260 kBq h/m ) than that recorded in the Vetralla
6 and figure 4 show the percentages of cells for both
experiment (1,090 kBq h/m 3 ).
Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison
between in utero and Neonatal Exposures
Table 5 Damage as assessed by the Comet assay. N = number of mice; n.a. = not available; significance respect to control: a = P < 0.05.
Tail moment Control (before exposure)
Number of cells
DNA damage (%)
6.40 ± 3.24 Exposure 200 [kBq h/m 3 ]
8.98 ± 4.99 Exposure 1,160 [kBq h/m 3 ]
16.29 ± 4.89 a 21 ± 13.86 a
Table 6 Percentages of cells with DNA damage absent, low, medium, high, and total; significance respect to control: c = P < 0.001.
% cells for Exp. 1,160 kBq % DNA damage
% cells for Exp. 200 kBq
h/m 3 h/m 3 No damage (< 5%)
% cells in Controls
32.1 34.04 28.64 Low damage (5 ÷ 20%)
54.32 56.98 33.57 Medium damage (20 ÷ 40%)
12.96 8.79 30.87 c High damage (40 ÷ 95%)
0.62 0.19 6.92 c Total damage (> 95%)
Fig. 4 Histogram of percentages of cells with DNA damage absent, low, medium, high, and total.
The difference observed between the two groups in These results confirm that concentrations of radon the tests could be due to the differing period of
decay products, such as bismuth and polonium (the exposure to radon and its decay products, indicating a
main radioactive isotopes emitting alpha particles) are higher degree of susceptibility during the postnatal
significantly lower in foetal tissues than in mothers period.
[18, 19]. Similarly, Kendall [20] has also shown that
74 Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison
between in utero and Neonatal Exposures
concentrations of radionuclides in embryonic tissues lower than those that cause genetic damage in adults are almost systematically lower than the 3 and embryos (about 1,000 kbq/m ). It would therefore
concentrations found in the tissues of the mother, and
be appropriate to carry out further studies on this issue, that the first part of a newborn’s life (breastfeeding) is
in order to propose the most suitable prevention
a period in which the absorbed dose is higher. strategies in terms of social-health. The same results were observed in the experiments
Indoor radon levels are affected by the soil performed with adult mice in the Vetralla cellar.
composition under and around the buildings, and by Exposure to radon emissions induces a significant
the easiness with which radon enters the houses. increase in micronuclei frequencies in peripheral
Homes that are very close to each other can have blood erythrocytes (ME/1,000E). Final exposure, after
different indoor radon levels. In addition, precipitation,
barometric pressure, climate change parameters, 2nd experiment: 1,160 kBq h/m 3 ) caused a hydrogeological phenomena and other factors can
more than thirty days (1st experiment: 1,700 kBq h/m 3 ;
statistically significantly higher frequency of ME than cause radon levels to vary from month to month or that observed in the control samples (taken before
day to day, which is why both short- and long- term exposure).
tests are available. The graphics showing the The results highlighted by the Micronucleus test
measurements of radon concentration registered in the were confirmed in the second experiment carried out
cellars can be a good illustration of the varying of the on adults, through the application of the Comet assay.
radon levels in a confined environment. However, This test, has, in fact underlined a statistically
exposure obtained in the two experiments, can be used significant increase in DNA damage after exposure to
to say that the area of Vetralla could due to higher radon than the damage detected in the control samples
concentrations. Of course it depends by the soil (taken before the exposure).
composition of the area.
4. Conclusions
5. Conflict of Interests
In conclusion, the work presented here confirms, on The authors declare no conflict of interests the basis of experimental evidence conducted on mice
regarding the publication of this paper. whose the biological model is perfectly comparable to
Acknowledgments
the human species as already observed in previous studies [21-25], the effects caused by prolonged
We would like to thank: Prof. Fabrizio Aumento exposure to radon gas emissions.
(Tuscia University, Viterbo), Doctor Massimo In particular, the present study aims to highlight
Esposito (“U-Series”, Bologna) for their technical that workers in certain subterranean environments
help and giving access to their facilities and (bank vaults, catacombs, etc.), individuals living in
instruments; D’Ambrosi, Marcuz and Megna families homes constructed of tufaceous material and those
for use of the Ciampino cellars and Di Carlo and who have settled in volcanic areas are all subject to
Gomeno families for the use of the Vetralla cellar; the potential genetic risks.
Research Group composed by Prof. Felipe Cortés, This study has shown a particular sensitiveness to
Santiago Mateos, Nuria Pastor and Paula Daza at the radon and its decay products that is not as strong in
Department of Cell Biology, Faculty of Biology, murine embryos, protected by the placental tissues but
University of Seville (Spain) for their technical help, appears in newborn mice, which already display
their warm welcome and access to their facilities too.
genetic damage from exposure levels (260 kbq/m 3 )
Thanks also to: Prof. Caterina Tanzarella and Dr.ssa
Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison
between in utero and Neonatal Exposures
Luisa Anna Ieradi for kind suggestions and helpful “Exposure of Red Bone Marrow to Ionising Radiation from Natural and Medical Sources in France.” Journal
instruction; Doctorate School of Industrial and
21: 209-19. Environmental Hygiene directed by Prof. I. [10] Hellmann, B., Friis, L., Vaghef, H., and Edling, C.
Radiological Protection
Figà-Talamanca; PRIN 2012 project “Effetti dei 1999. “Alkaline Single Cell Gel Electrophoresis and cambiamenti climatici sulle microteriocenosi terrestri”
Human Biomonitoring for Genotoxicity: a Study on Subjects with Residential Exposure to Radon.”
(Effects of climate change on terrestrial Mutation Research 442: 121-32.
microtheriocoenoses); Prof. P. Brandmayr. Thanks to [11] Fukutsu, K., Ya mada, Y., Zhuo, W., and Koizumi, A. Zach Butcher for the careful proof reading.
2005. “Induction of Micronuclei in Rat Tracheal Epithelial Cells Following Radon Exposure at
References
Air-liquid Interface Culture.” International Congress Series 1276: 251-3.
[1] UNSCEAR (United Nations Scientific Committee on [12] Udroiu, I., Cristaldi, M., Ieradi, L. A., Bedini, A., the Effects of the Atomic Radiation), “Sources and Giuliani, L., and Tanzarella, C. 2006. “Clastogenicity Effects of Ionizing Radiation,” United Nations, New
and Aneuploidy in Newborn and Adult Mice Exposed York, 2000. to 50 Hz Magnetic Fields.” International Journal of [2] Ruano-Ravina, A., Ruosteenoja, E., Schaffrath, R. A.,
82 (8): 561-7. Tirmarche, M., Tomáek, L., Whitley, E., Wichmann, H.
Radiation Biology
[13] Ieradi, L. A., Cristaldi, M., Ermenegildi, A., La E., and Doll, R. 2005. “Radon in Homes and Risk of
Barbera, L., Radicchi, L., Renzopaoli, F., Esposito, M., Lung Cancer: Collaborative Analysis of Individual
and Lombardi, S. 2008. “Mutagenic Effects in Mice Data from 13 European Case-control Studies.” British
Exposed to Radon-222 Emissions in Latium Region Medical Journal 330 (7485): 223.
(Italy).” Fresenius Environmental Bulletin 17 (9b): [3] Schuttmann, W. 1993. “Schneeberg Lung Disease and
1420-5.
Uranium Mining in the Saxon Ore Mountains (Erzg [14] Daza, P., Torreblanca, J., and Moreno, F. J. 2004. “The ebirge).” American Journal of Industrial Medicine 23
Comet Assay Differentiates Efficiently and Rapidly (2): 355-68.
between Genotoxins and Cytotoxins in Quiescent [4] Greenberg, M., and Selikoff, I. J. 1993. “Lung Cancer
Cells.” Cell Biology International 28: 497-502. in the Schneeberg Mines: a Reappraisal of the Data
[15] Singh, N. P., Mc Coy, M. T., Tice, R, R., and Report.” The Annals of Occupational Hygiene 37 (1):
Schneider, E. L. 1988. “A Simple Technique for 5-14.
Quantitation of Low Levels of DNA Damage in [5] IARC—International Agency for Research on Cancer,
Individual Cells.” Experimental Cell Research 175: “Man-made Mineral Fibres and Radon. IARC
184-91.
Monographs on the Evaluation of Carcinogenic Risks [16] Fairbairn, D. W., Olive, P. L., and O'Neill, K. L. 1995. to Humans 43.” IARC, Lyon, 1998.
“The Comet Assay: a Comprehensive Review.” [6] Darby, S. C., Whitely, E., Howe, G. R., Hutchings, S.
Mutation Research 339: 37-59. J., Kusiak, R. A., Lubin, J. H., Morrison, H. I.,
[17] Anderson, D., Yu, T. W., Phillips, B. J., and Schmezer, Tirmarche, M., Tomásek, L., Radford, E. P., Roscoe, R.
P. 1994. “The Effect of Various Antioxidants and J., Samet, M. J., and Yao, S. X. 1995. “Radon and
Other Modifying Agents on Oxygen-radical-generated Cancers Other Than Lung Cancer in Underground
DNA Damage in Human Lymphocytes in the COMET Miners: a Collaborative Analysis of 11 Studies.”
Assay.” Mutation Research 307: 261-71. Journal of the National Cancer Institute 87 (5):
[18] ICRP, “Doses to the Embryos and Fetus from Intakes 378-84.
of Radionuclides by the Mother. ICRP Publicationn [7] Nelson, S. L., and Grosovsky, A. J. 1995. “Evidence
88.” Annals of the ICRP 31 (1-3) (Oxford Elsevier for a Gene Distal to HPRT which Restricts the
Science), 2001.
Recovery of Large Deletions.” Environmental [19] Stather, J. W. 2003. “Dose Coefficients for the Embryo Molecular Mutagenesis 25: 39-43.
and Foetus following Intakes of Radionuclides by the [8] Schlegel, R., and Mac Gregor, J. T. 1982. “The
Mother.” Journal of Radiological Protection 22: 7-24. Persistence of Micronuclei in Peripheral Blood
[20] Kendall, G. M., Fell, T. P., and Harrison, J. D. 2009. Erythrocytes: Detection of Chronic Chromosome
“Dose to Red Bone Marrow of Infants, Children and Breakage in Mice.” Mutation Research 104: 367-9.
Adults from Radiation of Natural Origin.” Journal of [9] Rommens, C. C., Ringeard, C., and Hubert, P. 2001.
Radiological Protection
29: 123-38.
76 Genotoxic Effects in Mice Exposed to Radon Emissions in Indoor Conditions. Comparison between in utero and Neonatal Exposures
[21] Little, C. C. 1937. “US Science Wars against an
420: 520-62.
Unknown Enemy: Cancer.” Life 2: 11-7. [24] Paigen, K. 2003. “One Hundred Years of Mouse [22] MacKey, B. E., and Mac Gregor, J. T. 1979. “The
Genetics: an Intellectual History. I. The Classical Micronucleus Test: Statistical Design and Analysis.”
Period (1902-1980).” Genetics 163: 1-7. Mutation Research 64 (3): 195-204.
[25] Paigen, K. 2003. “One Hundred Years of Mouse [23] Waterston, R. H., et al. 2002. “Initial Sequencing and
Genetics: an Intellectual History. II. The Molecular Comparative Analysis of the Mouse Genome.” Nature
Revolution (1981-2002).” Genetics 163: 1227-35.
Journal of Life Sciences 10 (2016) 77-84
doi: 10.17265/1934-7391/2016.02.003
DAVID PUBLISHING
Stochastic Lattice gas Cellular Automata Model for Epidemics
Ariel Félix Gualtieri and Juan Pedro Hecht Department of Biophysics, Faculty of Dentistry, University of Buenos Aires, Buenos Aires C1122AAH, Argentina
Abstract: The aim of this study was to develop and explore a stochastic lattice gas cellular automata (LGCA) model for epidemics. A computer program was development in order to implement the model. An irregular grid of cells was used. A susceptible-infected-recovered (SIR) scheme was represented. Stochasticity was generated by Monte Carlo method. Dynamics of model was explored by numerical simulations. Model achieves to represent the typical SIR prevalence curve. Performed simulations also show how infection, mobility and distribution of infected individuals may influence the dynamics of propagation. This simple theoretical model might be a basis for developing more realistic designs.