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From the School of Optometry, University of Waterloo, Ontario, Canada.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Aseptically dissected porcine lenses were cultured in glass chambers. At 1 week, lenses were exposed to a predetermined UV energy (J/cm2) at specific wavebands ranging from 270 to 370 nm at 5- and 10-nm intervals. The UV energy was generated by a PRA integrated arc lamp system using a water-cooled 1000 W, high-pressure xenon lamp. The lamp output was limited using a deionized water filter, a monochromator, and secondary optics. An electronic shutter was used to control the exposure time. The median effective dose, ED50 (i.e., UV energy threshold) for each waveband was statistically determined using probit analysis. Irradiated spots (3.06 mm2) on the lenses were monitored every 6 to 12 hours up to 48 hours postirradiation for any UV-induced opacity with a dissecting microscope and photomicrography. The ED50s were plotted against wavelengths to obtain the action spectrum.
RESULTS. The threshold values for 270, 300, and 365 nm were 0.057, 0.069, and 137.19 J/cm2, respectively. Permanent UV-induced cataract was obtained at twice the threshold values for UVB and UVA.
CONCLUSIONS. An action spectrum for in vitro UV-induced cataract using whole cultured lens is established. These data are comparable to published in vitro (with isolated lens epithelial cells) and in vivo action spectra. The recovery pattern appears to be similar to the in vivo situation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12% higher than they
were during similar periods earlier in the decade. Their report
provides the strongest evidence yet that degradation of the
stratospheric ozone layer is ongoing and produces more hazardous UV
exposure conditions for life on the Earth’s surface.7
It
is evident that stratospheric ozone thinning is occurring not just in
the sparsely populated polar regions, but also above populous
midlatitude regions such as northern Europe, Canada, New Zealand, and
Australia.7 In the eye, it is believed that UV photochemical damage occurring in the crystalline lens can persist for a long time, causing temporary or permanent impairment of vision. The specific targets and the detailed mechanisms that are responsible for UV-induced cataracts remain to be determined.17 18 19 20 21 An important aspect of solar UV radiation investigation is the UV cataractogenic action spectrum in the wavebands to which humans are exposed. In terms of UV-lens research, the in vivo UV action spectrum by Bachem22 and Pitts et al.23 24 and the in vitro UV action spectrum for lens epithelial cells by Andley et al.21 and Andley and Weber25 are available in the literature. However, in vitro UV action spectrum data for the cultured intact whole lens are lacking. Because many experimental models now focus on the intact cultured whole lens and because the cellular targets and mechanisms of action of UV radiation vary as a function of wavelength, it is necessary to establish an in vitro UV action spectrum for the cultured whole crystalline lens. From the action spectrum, the mechanisms of UV effects within each waveband can be effectively studied.
The action spectrum of a biological response is the variation in magnitude of the response with wavelength. The shape of the resulting curve is related to the absorption of the radiation-absorbing molecule initiating the response. The present study determined the action spectrum for in vitro UVR cataract formation (gross superficial opacities) using organ cultured whole porcine crystalline lens. Pitts et al.23 24 developed the data on the crystalline lens in vivo UV action spectrum for wavelengths from 295 to 365 nm. These data are still the best existing action spectrum data for in vivo UV cataractogenesis.26 The effort to reduce or eliminate the use of live animals in biomedical research is now shifting experimental approaches from in vivo to in vitro. The data of Pitts et al.23 24 have been used for some time for most in vivo UV experimental applications. More recently, a series of experiments on in vitro UV action spectra for isolated lens epithelial cells has been reported by Andley et al.21 Their data are plausible, but it is uncertain whether or not the data are directly applicable to studies involving the whole intact lens. Thus, the purpose of the present study was to determine the in vitro action spectrum for UV lenticular damage in cultured whole lens and to study the damage recovery pattern. Because the pig’s embryological growth and development are typical of mammals and its shape and size are similar to the human lens, some inferences may be made to the human lens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 mm remained on top of the anterior surface of
the lens. The lens exposures to different UV radiant energy levels at
the defined wavelengths were carried out at room temperature.
UVR Exposure System
UV energy was generated from a Photochemical Research Associates
(PRA) integrated arc lamp system using a water cooled 1000 W high
pressure xenon arc (Photochemical Research Associates Inc., London,
Ontario, Canada). The source for UVB and UVA radiation was controlled
by a PRA lamp power supply (model 301), which is water-cooled for
optimal stability. In the power supply, provision is made for automatic
shutdown if the supply temperature exceeds preset limits in case of
coolant failure. An automatic reset resumes power supply operation when
safe temperature limits are re-established. Figure 1
illustrates the UV irradiation setup and the lens incubation chamber.
The infrared (IR) output was absorbed by a 9-cm-long quartz-enclosed
deionized distilled water chamber placed between the arc source exit
and a quartz condensing lens. The quartz condensing lens, 5 cm in
diameter placed between the water chamber and monochromator, focused
the UV radiation on the entrance slit of the monochromator.
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To convert the radiometer measurement to irradiance, the measured value
was multiplied by the irradiated area on the lens and the linear
multiplication factor from the calibration curve. The irradiated area
at the lens surface was 3.06 mm2 (0.0306
cm2). The reciprocal of 0.0306 equals 32.68;
thus, the unit area multiplication factor for conversion to irradiance
was 32.68 throughout this experiment. For example, the linear
multiplication factor for 300 nm was 1.08; therefore, a reading of 48
µW would be equal to 1694 (i.e., 48 x 1.08 x 32.68)
µW/cm2 as irradiance. Radiant exposure time was
determined using the following radiometric equation: t
H/E
, where t
is exposure duration (seconds), H is radiant exposure
(J/cm2), and
E is measured irradiance
(W/cm2). The duration of exposure was controlled
by a preset electronic counter, which automatically closed the shutter
after each predetermined exposure. The shutter system allowed the
control of exposure duration to any length. In this study exposure time
ranged from 49 seconds to 22 hours.
Exposure Sequence and Assessment
A modified staircase (up-and-down) method28
with
doubling, 50%, 25%, and 10% decrement/increment steps, was used to
obtain threshold values for each waveband from 270 to 370 nm. On
average, five lenses were irradiated for each energy level at each
waveband. If none of the five lenses at a given exposure level showed
damage, then the dose was doubled. If all five lenses showed damage,
the dose was reduced by 50%. If three of five lenses did not show
damage, the energy level was arbitrarily increased by 25% or 10% of
the immediate previous dose. This was continued until at least three of
five lenses were observed to show damage before proceeding to the next
energy level or waveband. All lenses were examined under a dissecting
light microscope to determine their suitability for experimentation.
The pig lenses could be kept viable in culture medium for 5 to 6
weeks.29
The UV beam was focused at the anterior pole for
all the lenses to keep the irradiated area the same.
For the purpose of deciding on the starting point, the threshold data of Pitts et al.23 24 were used. They found that the most efficient waveband for lenticular damage was 300 nm, which had a radiant exposure threshold of 0.15 J/cm2. An approximate UV energy level of 0.2 J/cm2 at 300 nm was chosen as the starting point in the present study. Therefore, the first exposure was made at 300 nm, with 0.2 J/cm2 delivered to the anterior lens surface. The sequence involved irradiation for wavelengths from 300 to 270 nm in 5-nm intervals and then 305 to 370 nm in 5- or 10-nm intervals. Observation time was limited to a maximum of 36 to 48 hours after irradiation. Irradiated spots on the lenses were monitored every 6 to 12 hours for any morphologic lesions (i.e., superficial or subcapsular opacities), induction time, and lesion pattern with photomicrography (Nikon dissecting microscope, Tokyo, Japan). During photomicrography, the eyepiece magnification was set at x10 and microscope magnification at x2.5.
Photographs of the lesions patterns (square-shaped area of 3.06 mm2) were randomly taken at appearance or during recovery. UV-irradiated lenses were visually compared with untreated controls. Again, if no lesion was found at 36 to 48 hours, the dose was doubled or increased by 50%, 25%, or 10% for the next exposure. If there was a lesion with the increase, the dose was arbitrarily decreased by 25% or 10% until there was no lesion. For each subsequent set of lenses, the dose was decreased or increased, depending on the response of the previous set of lenses to a lower radiant exposure, until moderate or severe lesions were observed in at least three lenses of five. The ED50 (i.e., 50% probability of damage, with 95% confidence interval) was then calculated by using probit analysis.28 An SPSS for Windows software (SPSS Inc., Chicago, IL)30 was used for the probit analysis. As an example, Table 1 shows the results from the probit analysis of the 300-nm waveband data. The induction time and the number of lenses used per radiant exposure were recorded. ED50 was determined for wavebands centered on 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 330, 340, 350, 360, 365, and 370 nm, and values are presented in Table 2 . The 365-nm waveband was included because most studies on UVA effects center on this wavelength. The ED50 values were plotted as a function of wavelength to determine the action spectrum curve (Fig. 2) .
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Damage Grading
The severity of the lesions in the lenses was graded as follows:
-, no damage; +, moderate or threshold damage, lesions half or less of
square pattern; + +, severe damage, lesions fill more than half of
square pattern; + -, lesions disappearing (sign of recovery); and
- -, lesions completely disappeared (full recovery).
Any lens having a + means that the lens showed damage at that particular energy level (dose). For analysis, a lens with + or + + grade is counted with the number of lenses damaged. Any lens with a lesion showing recovery was graded + -, and the time that the repair trend was observed was recorded. If a lens lesion showed complete recovery, it was graded - -, and the time of the observation noted. Any lens with a damaged capsule was discarded. The control lenses were also observed for any morphologic changes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Graphically, a threshold can be obtained by connecting the 50% probability on the y-axis to the corresponding radiant exposure value on the x-axis through the regression line intercept. The different ED50 (i.e., 50% probability of damage) values for the wavebands were collated and plotted against wavelengths to obtain the action spectrum (see Fig. 2 ). The data generally indicate shorter damage latency and slower recovery for higher UV energy levels, whereas the reverse is the case for lower energy levels. It should be noted that at 370 nm, the highest dose that could practically be given produced no visually observable lesion. The contribution of the adjacent wavelengths was considered to be relatively negligible. The summary of the probit analysis for other wavebands are reported elsewhere.27
Part 2
With respect to damage reversibility, group 1 lenses showed
lesions at 36 to 48 hours postexposure and complete recovery after 10
days (Fig. 3)
. In group 2 lenses, lesions were visible at 24 hours, with no complete
recovery occurring at week 4 after exposure (Fig. 4)
. The lesions in group 3 lenses appeared at 12 to 24 hours, 50% of
the lesions persisted in all lenses at least 4 weeks postexposure (not
shown). Among group 4 lenses, lesions were visible at 24 hours
postexposure, with 50% of the lesions persisting up to the 4 weeks
of study (Fig. 5)
. It should be noted that in Figures 3
4
and 5
, all the photographs
for each figure were of the same lens.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The in vitro action spectrum obtained in the present study shows a trend similar to the in vivo data of Pitts et al.23 that extended from 295 to 395 nm. Because of the absence of corneal absorption, it was possible to induce lenticular opacity with shorter wavelengths down to 270 nm in the present study. This was done in the hope that the obtained threshold (ED50) values could be useful for future in vitro UV lens toxicology investigations extending below 290 to 270 nm. Therefore, the data in the present study involved wavelengths from 270 to 370 nm. The limit at the longer UV wavelengths was at 370 nm in the present study because no observable damage could be induced with the highest radiant exposures at the 370-nm waveband. This tends to agree with Andley et al.,21 who reported that the highest dose at the 405-nm waveband in their study produced no adverse effect on lens epithelial cells. Also, using 162 J/cm2 radiant exposure at 365-nm waveband, which was the highest dose in their study, Pitts et al.23 could not achieve any lenticular opacities in vivo in rabbit.
By comparison, at 300 nm, the present study found 0.069
J/cm2 as threshold, whereas Pitts et
al.23
found 0.15 J/cm2 for in vivo
exposure. Andley et al.21
and Andley and
Weber25
found 0.068 and 0.052 J/cm2
at 302 nm, respectively, using cultured rabbit and human lens
epithelial cells. Data in the present study showed radiation at 295-nm
waveband to be 25 times more effective than 315-nm radiation in
producing UV-induced lens anterior subcapsular lesions (Table 2)
,
whereas the data of Pitts et al.23
showed that 295-nm
radiation was six times more effective than 315-nm radiation. The data
of Andley et al.21
and Andley and Weber25
show that the radiation at 297 nm is 171 and 261 times more effective
than the 313-nm radiation used in their respective studies with rabbit
and human lens epithelial cells. The difference in relative effectivity
is not surprising because of the absence of corneal and aqueous
absorption in the in vitro conditions. Moreover, in isolated epithelial
cells, the influence of overlying capsule, underlying cortex, and
adjacent cells are absent or minimal with respect to the impact of the
radiation. The in vitro radiant exposure values in the present study
are 29 times lower at 295 nm, 2 times lower at 300 nm, 3 times at
305 nm, 3 times at 310 nm, 6 times at 315 nm, and 4 times at 320
nm than in vivo data reported by Pitts et al.23
for
UV-induced lens opacities in rabbits. Beginning from 330 nm, both
the in vitro and in vivo thresholds are similarly high.
The data in the present study showed that very high radiant exposure is
needed from wavelengths longer than 325 nm to induce subcapsular
lesions in vitro; this is in agreement with Pitts,17
who
mentioned that radiant exposures above 320 nm need to be quite high to
have an effect during in vivo experiments. The increased radiant
exposure required at longer wavelengths to produce lenticular damage is
not surprising because of the lower relative effectiveness of longer UV
wavelengths. Although end points used in the present study were
different from those in the studies of Pitts et al.,23
24
Andley et al.,21
and Andley and Weber,25
results of the present study indicate that the wavelengths of UVR that
are most effective at inducing cataractous changes in the crystalline
lens in vivo23
24
are similar. The in vitro action
spectrum in the present study begins at 270 nm and extends to 365
nm, whereas the in vivo action spectrum begins at 295 nm and extends to
335 nm.24
Pitts et al.24
found the lens
and corneal curves to be relatively parallel from 300 nm up to 320
nm. The present study found the threshold radiant exposure at 300- and
305-nm wavebands to be similar to the values reported for corneal
damage. This similarity between lenticular and corneal UV thresholds is
not surprising because the lens is a cellular structure consisting of
epithelial cells from the same germinal source (surface ectoderm) as
that of the corneal epithelium.
The relatively low radiant exposures required to produce damage in the 270- to 315-nm wavelength range suggest that the most effective waveband for producing UV cataract in vitro is from 270 to 315 nm. Looking at the trend of the action spectrum (Fig. 2) , it would be expected that wavelengths shorter than 285 nm should be more effective (i.e., have lower thresholds because the shorter the UV wavelength, the higher the photon energy). However, there was a fair rise in effective dose below 285 nm (Fig. 2) , which suggests that a different mechanism is responsible for UVR lenticular toxicity below 285 nm. Another possible explanation is that UVR does not affect crystalline lens biomolecules in the same way for each waveband.17 The action spectrum curve shows that 285 nm is the most effective wavelength for producing UV toxicity on porcine lens in vitro. This is relatively close to 280 nm, the shorter absorption maximum of the human crystalline lens that has been reported to exhibit absorption maxima at 280 and 370 nm.31 32 33 With the advent of the artificial cornea, the maximum effectivity at the 285-nm wavelength on the lens is important. The present in vitro result support Yamanashi et al.,34 who explained that the wavelength at which UV-induced cataract appears to occur maximally (i.e., 300 nm) due to corneal absorption during an in vivo situation might not be the true maximum of UV-induced cataractogenesis. At the long UV wavelengths, 365 nm appears to be more effective than 360 nm for causing cataract. The present study has established the in vitro UV action spectrum for cultured porcine lens using gross morphologic changes (anterior subcapsular opacities) as damage criteria. However, extrapolations from the data can be applied to the study of in vitro UV-induced cataracts using lenses from other animal species. The UV levels in this study was estimated to be approximately nine times higher than ambient lenticular levels reported by Zigman.35
In terms of recovery from UV damage, the present study confirms the findings by Pitts et al.23 24 that radiant exposure at two times threshold level results in permanent opacity. At two times UVA threshold exposures, there was no full recovery (Fig. 5) , confirming that UVA is cataractogenic. At five times UVB threshold exposure, the photodamage gave rise to prominence of the suture, which might be an indication of permanent opacity with no chance of recovery. Data in the present study support the theory that repair will generally occur for UVR-induced cataract at threshold and subthreshold radiant energy levels. This may tempt one to speculate that the temporary blurring of vision often experienced due to UVR-induced photokeratoconjunctivitis may arise from not only the cornea but also from the lens.
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Acknowledgements |
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Footnotes |
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Submitted for publication April 9, 2001; revised June 8, 2001; accepted June 13, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Olanrewaju M. Oriowo, School of Optometry, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada. loriowo{at}sciborg.uwaterloo.ca
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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