Cytogenetic, cellular, and developmental responses in ...teristics and molecular responses of...
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Cytogenetic, cellular, and developmental responses in antarctic sea urchins (Sterechinus neumayeri) following laboratory ultraviolet-B and ambient solar radiation exposures SUSAN ANDERSON, JENNIFER HOFFMAN, and GILLIAN WILD, Lawrence Berkeley Laboratory, Berkeley, California 94720 ISIDRO BOSCH, Department of Biology, State University of New York, Geneseo, New York 14454 DENEB KARENTZ, Department of Biology, University of San Francisco, San Francisco, California 94117-1080 T here is widespread concern that increasing ultraviolet-B (T.JV-B) radiation [280-320 nanometers (nm)] as a conse- quence of springtime ozone depletion could harm antarctic ecosystems. Yet efforts to evaluate potential detrimental effects have been relatively limited, focusing largely on poten- tial alterations in phytoplankton production (Helbling et al. 1992; Smith et al. 1992). Little has been done to evaluate effects on animal populations. To our knowledge, there have been no attempts to evaluate the potential DNA damage (genotoxic damage) that may occur as a consequence of heightened UV-B exposure. Several techniques for studying genotoxic effects are available and have been used to eluci- date the effects of radiation and chemicals in aquatic ecosys- tems (Shugart et al. 1992). Our goal was to apply some of these techniques to evaluate UV-B effects in marine animals of Antarctica. We developed an aquatic animal model that can be used to evaluate genotoxic, cellular, and developmental responses to UV-B exposure using embryos of the sea urchin Sterechinus neumayeri. Embryos were exposed either to ambient solar radiation or to UV-B lamps in the laboratory. All studies were performed at Palmer Station, Antarctica. Embryos were sam- pled at varying developmental stages and preserved in forma- un for both cytogenetic analysis and scoring of developmental abnormalities. The laboratory and in situ experiments were designed to address two goals. The first was to determine whether the selected genotoxic and cellular responses would exhibit dose-effect relationships in response to UV-B exposure. The second was to determine whether genotoxic and/or cellular Daily average values of integrated incident UV-B radiation (calculated from hourly scans) and concentrations of stratospheric ozone over Palmer Station, Antarctica. Data obtained from the National Science Foundation UVMonitoringNetwork. 16 November 1991976 301 17 November 1991 1,344 282 18 November 1991 2,114 304 19 November 1991 1,804 296 a ln microwatts per square centimeter per second. bin Dobson units. responses could be observed after exposure to in situ levels of UV-B. The laboratory dose-effect study involved exposing embryos in four age groups. Embryo cultures were derived from animals obtained from both shallow [less than 3 meters (in)] and deep (greater than 20 m) populations of urchins. Separate experiments on these two populations were initiated using two- to eight-cell-stage embryos; three additional age groups were exposed 24, 48, and 96 hours (h) following the initial timepoint. Following exposure, embryos were held for 24 h in glass vials before fixation. Exposures were delivered using 12-inch fluorescent lamps (BLE-8TB02, Spectronics, Corp., Westbury, New York), according to the general meth- ods of Karentz, Cleaver, and Mitchell (1991). Exposures were to 0, 10, 100, and 1,000 joules per square meter (Jim2). In the in situ experiment, embryos were exposed at four depths (1, 3, 5, and 7 m) in Whirlpak bags with and without mylar for UV-B filtration. All exposures were conducted in triplicate and initiated with approximately 2,000 two- to eight-cell embryos per bag. Only urchins from shallow popu- lations were used to establish the embryo cultures. Whirlpak bags were secured to a buoyed rack (Karentz and Lutze 1990) in 20 in of water off Palmer Station. The in situ experiment was conducted during the period 16-19 November 1991, during which ozone column readings ranged from 282 to 304 Dobson units (DU), and hourly scans of incident UV-B averaged from 976 to 2,114 microwatts per square centimeter per second (tW/cm 2 /sec) daily (table). Weather during the experimental period was variable. On the first day, open water and overcast conditions prevailed. Day 2 was partly sunny, and on day 3, the experimental area was covered by pack ice and overcast conditions prevailed. Formahin-preserved embryos were analyzed for cytoge- netic and cytologic alterations according to the general meth- ods of Hose and Puffer (1983), with extensive modification for use with S. neumayeri. Briefly, following postfixation in 45 percent acetic acid for 15 minutes, embryos were stained with an aceto-orcein solution composed of 19 parts of standard aceto-orcein with one part propionic acid. Twenty embryos for each treatment were examined for anaphase aberrations including attached fragments, acentric fragments, transloca- tion bridges, and unequal chromosome distributions. Cyto- logic abnormalities, including such conditions as abortive mitoses, pycnosis, and karyolysis, were also examined. Preliminary data from the laboratory study (embryos derived from the shallow population and sampled at 24 h after the two- to eight-cell stage) indicate that anaphase aber- ration frequencies in embryos increase in a dose-dependent ANTARCTIC JOURNAL - REVIEW 1993 115
Transcript of Cytogenetic, cellular, and developmental responses in ...teristics and molecular responses of...
Cytogenetic, cellular, and developmental responses in antarcticsea urchins (Sterechinus neumayeri) following laboratory
ultraviolet-B and ambient solar radiation exposuresSUSAN ANDERSON, JENNIFER HOFFMAN, and GILLIAN WILD, Lawrence Berkeley Laboratory, Berkeley, California 94720
ISIDRO BOSCH, Department of Biology, State University of New York, Geneseo, New York 14454DENEB KARENTZ, Department of Biology, University of San Francisco, San Francisco, California 94117-1080
There is widespread concern that increasing ultraviolet-B(T.JV-B) radiation [280-320 nanometers (nm)] as a conse-
quence of springtime ozone depletion could harm antarcticecosystems. Yet efforts to evaluate potential detrimentaleffects have been relatively limited, focusing largely on poten-tial alterations in phytoplankton production (Helbling et al.1992; Smith et al. 1992). Little has been done to evaluateeffects on animal populations. To our knowledge, there havebeen no attempts to evaluate the potential DNA damage(genotoxic damage) that may occur as a consequence ofheightened UV-B exposure. Several techniques for studyinggenotoxic effects are available and have been used to eluci-date the effects of radiation and chemicals in aquatic ecosys-tems (Shugart et al. 1992). Our goal was to apply some ofthese techniques to evaluate UV-B effects in marine animalsof Antarctica.
We developed an aquatic animal model that can be usedto evaluate genotoxic, cellular, and developmental responsesto UV-B exposure using embryos of the sea urchin Sterechinusneumayeri. Embryos were exposed either to ambient solarradiation or to UV-B lamps in the laboratory. All studies wereperformed at Palmer Station, Antarctica. Embryos were sam-pled at varying developmental stages and preserved in forma-un for both cytogenetic analysis and scoring of developmentalabnormalities.
The laboratory and in situ experiments were designed toaddress two goals. The first was to determine whether theselected genotoxic and cellular responses would exhibitdose-effect relationships in response to UV-B exposure. Thesecond was to determine whether genotoxic and/or cellular
Daily average values of integrated incident UV-Bradiation (calculated from hourly scans) andconcentrations of stratospheric ozone over PalmerStation, Antarctica. Data obtained from the NationalScience Foundation UVMonitoringNetwork.
16 November 1991976 30117 November 1991 1,344 28218 November 19912,114 30419 November 1991 1,804 296aln microwatts per square centimeter per second.bin Dobson units.
responses could be observed after exposure to in situ levels ofUV-B. The laboratory dose-effect study involved exposingembryos in four age groups. Embryo cultures were derivedfrom animals obtained from both shallow [less than 3 meters(in)] and deep (greater than 20 m) populations of urchins.Separate experiments on these two populations were initiatedusing two- to eight-cell-stage embryos; three additional agegroups were exposed 24, 48, and 96 hours (h) following theinitial timepoint. Following exposure, embryos were held for24 h in glass vials before fixation. Exposures were deliveredusing 12-inch fluorescent lamps (BLE-8TB02, Spectronics,Corp., Westbury, New York), according to the general meth-ods of Karentz, Cleaver, and Mitchell (1991). Exposures wereto 0, 10, 100, and 1,000 joules per square meter (Jim2).
In the in situ experiment, embryos were exposed at fourdepths (1, 3, 5, and 7 m) in Whirlpak bags with and withoutmylar for UV-B filtration. All exposures were conducted intriplicate and initiated with approximately 2,000 two- toeight-cell embryos per bag. Only urchins from shallow popu-lations were used to establish the embryo cultures. Whirlpakbags were secured to a buoyed rack (Karentz and Lutze 1990)in 20 in of water off Palmer Station.
The in situ experiment was conducted during the period16-19 November 1991, during which ozone column readingsranged from 282 to 304 Dobson units (DU), and hourly scansof incident UV-B averaged from 976 to 2,114 microwatts persquare centimeter per second (tW/cm 2 /sec) daily (table).Weather during the experimental period was variable. On thefirst day, open water and overcast conditions prevailed. Day 2was partly sunny, and on day 3, the experimental area wascovered by pack ice and overcast conditions prevailed.
Formahin-preserved embryos were analyzed for cytoge-netic and cytologic alterations according to the general meth-ods of Hose and Puffer (1983), with extensive modification foruse with S. neumayeri. Briefly, following postfixation in 45percent acetic acid for 15 minutes, embryos were stained withan aceto-orcein solution composed of 19 parts of standardaceto-orcein with one part propionic acid. Twenty embryosfor each treatment were examined for anaphase aberrationsincluding attached fragments, acentric fragments, transloca-tion bridges, and unequal chromosome distributions. Cyto-logic abnormalities, including such conditions as abortivemitoses, pycnosis, and karyolysis, were also examined.
Preliminary data from the laboratory study (embryosderived from the shallow population and sampled at 24 hafter the two- to eight-cell stage) indicate that anaphase aber-ration frequencies in embryos increase in a dose-dependent
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Figure 1. Dose-effect relationships for sea urchin embryos (S.neumayeri) in laboratory exposure experiments.
manner in response to UV-B exposure, and in parallel, themean number of cells per embryo decreases (figure 1). Varia-tions in this pattern among shallow and deep populationsand among timepoints are still under investigation. Develop-mental abnormalities also increased in a dose-dependentmanner for embryos exposed at the two- to eight-cell stage.
The in situ experiments indicated that cytogenetic, cellu-lar, and developmental responses are all observable inembryos exposed at relatively shallow depths under ambientlight conditions. Embryos exposed at 1-m depths, both withand without mylar, exhibited 80 percent and 94 percentabortive mitoses, respectively; yet embryos exposed at 7-mdepths exhibited a low incidence of abortive mitoses in boththe UV-B transparent and mylar treatments (2 percent and 9percent, respectively). In addition, the mean number of cellsper embryo was much lower in the embryos exposed at 1 mthan in the embryos exposed at 7 m (figure 2). Data also indi-cate that normal development is inhibited at 1 m but not at 7m. Further examination of samples at the 3 m and 5 m depthsis underway.
We conclude that evaluation of cytogenetic, cellular, anddevelopmental alterations in sea urchin (S. neumayeri)embryos can be used to evaluate the potential detrimentaleffects of increasing UV-B in antarctic ecosystems. Subse-quent experiments should include an evaluation of theseparameters at several timepoints corresponding to varyinglevels of ozone depletion. Further research should also inves-tigate the kinetics of the response parameters, differentialeffects of UV-A and UV-B in ambient light, and extinctioncoefficients for the various responses.
We thank T. Gast, M. Slattery, and J. Hose for their assis-tance in this work. This research was supported by National
Figure 2. Cellular responses of sea urchin embryos (S. neumayeri) tofiltered and unfiltered ambient light exposures at two depths.
Science Foundation grant OPP 90-17664 to D. Karentz and anaward to S. Anderson from the Pew Scholars Program in Con-servation and the Environment. Development of the cytoge-netic techniques (S. Anderson) was supported under the Uni-versity of California Berkeley Superfund Basic Research Pro-gram under a contract through the U.S. Department of Energy(No. DE-ACO3-76SF00098).
References
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Hose, J.E., and H.W. Puffer. 1983. Cytologic and cytogenetic anom-alies induced in purple sea urchin embryos (Strongylocentrotuspurpuratus S.) by parental exposure to benzo(a)pyrene. MarineBiology Letters, 4, 87-95.
Karentz, D., J.E. Cleaver, and D. Mitchell. 1991. Cell survival charac-teristics and molecular responses of antarctic phytoplankton toultraviolet-B radiation. Journal of Phycology, 27(3), 326-341.
Karentz, D., and L.H. Lutze. 1990. Evaluation of biologically harmfulultraviolet radiation in Antarctica with a biological dosimeterdesigned for aquatic environments. Limnology and Oceanography,35(3),549-561.
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