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Glutathione depletion associated with rose bengal-photosensitized mortality in the housefly Musca domestica.

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Archives of Insect Biochemistry and Physiology 5:245-254 (1987)
Glutathione Depletion Associated With
Rose Bengal-Photosensitized Mortality in the
Housefly, Musca domestica
John M. Wages, Jr. and James R. Heitz
Deparfment of Biochemistry, Mississippi State University, Mississippi State, Mississippi
Glutathione, pyridine nucleotides, and lipid peroxides were measured in
adult houseflies following various regimens of dye treatment and light
exposure. Comparisons were made between dark control and light control
flies to judge the effect of light exposure alone; between dark control and
dark, dye-treated flies to evaluate the effects of dye-feeding in the dark; and
between dark, dye-treated and light, dye-treated flies t o measure the effect
of photodynamic action. N o significant effect was observed in levels of
NADC, NADH, or NADPC. However, a decrease (-16.7%) in NADPH during
photodynamic treatment was measured. Relatively small inductions of
glutathione were observed in light controls and dark, dye-treated flies.
Depletion of both CSH and total glutathione (the sum of GSH and GSSG,
expressed as GSH equivalents) occurred in light, dye-treated flies as compared
t o dark, dye-treated flies. Depletion of NADPH, when related t o GSH
depletion, suggested that GSH is being utilized to conjugate some products
of photooxidation or that it is being directly oxidized t o GSSG. However, the
observation of a reduction in total glutathione also suggests that a fraction of
GSH is being either oxidized t o a product other than GSSG or irreversibly
conjugated. No significant effects from photodynamic treatment o n
peroxidative potential or lipid hydroperoxides were observed.
Key words: lipid hydroperoxidation, photodynamic action, pyridine nucleotides
Halogenated derivatives of fluorescein (eg, rose bengal and erythrosin B)
have been shown to elicit mortality in a wide range of insect species [l].
Acknowledgments: This research was supported by funds from the Mississippi Agricultural
and Forestry Experiment Station (MAFES). The authors are indebted to Dr. Fay Hagan of the
Department of Experimental Statistics, MAFES, for her assistance in performing the statistical
analysis and to Drs. Marvin Salin, Maurice Kennedy, and Sonny Ramaswamy for their critical
review of the manuscript. This is MAFES publication No. 6350.
Received July 10,1986; accepted March 16,1987.
Address reprint requests to Dr. James R. Heitz, Department of Biochemistry, Mississippi State
University, P. 0. Drawer BB, Mississippi State, MS 39762.
01987 Alan R. Liss, Inc.
Wages and Heitz
Molecular mechanisms of photosensitization have been extensively reviewed
[2,3]. Xanthene dyes are believed to generate singlet oxygen [4] and also to
be capable of free radical-type reactions [5]. Although the literature abounds
with toxicological [6] and photochemical [7-91 investigations, relatively few
studies have been performed on intact organisms to determine the mechanism of photodynamic action.
Previously, Callaham et al.UO] observed that newly emerged boll weevils
(Anthonornus grundis B.), when fed a diet containing rose bengal, exhibited
no net weight gain or net protein synthesis, whereas total lipid levels decreased by greater than 50%. These results were interpreted as showing
metabolic energy depletion in the insect. Perturbation of the cellular energy
charge as reflected in the balance between the oxidized and reduced forms
of NADf and NADP+ could result in such an energy stress.
The tripeptide GSH is used in vivo as a cellular reservoir of cysteine, as a
radiation-protective antioxidant, in maintenance of enzymic thiol groups,
and in the detoxification of pesticides, which contributes to development of
resistance [ll]. Both free cysteine and the cysteinyl residue in GSH have
been shown to be directly photooxidized in vitro by various dyes [12,13]. In
addition, oxidation of sulfhydryl moieties of enzymes might be reflected in
GSH levels as a measure of thio1:disulfide status. Such an effect on enzymes
could be disastrous to the insect, decreasing the efficiencies of metabolic
Photosensitized hydroperoxidation of membrane lipids was suggested to
be a major biological effect of singlet oxygen by experiments with photooxidation of erythrocyte [14,15] and chloroplast [16] membrane lipids. Hydroperoxidation of membrane lipids in midgut epithelial cells would be expected
to lead to increased fragility and eventual lysis, which has been observed in
insects [17,18].
GSH, pyridine nucleotides, and unsaturated lipids were deemed likely
targets for the reactive products of photosensitization. The work reported
here was undertaken to determine whether photodynamic action affects
levels or redox states of these potential targets.
The houseflies (Muscu dornesticu L.) used in this study were a susceptible
strain maintained in the Department of Biochemistry, Mississippi State University [19]. Adult insects were not sexed but were used in the proportions
of males to females as emerged. Photodynamic treatments were performed
on flies less than 24 h posteclosion.
Photodynamic treatment was by exposure to General Electric 40 W Cool
White fluorescent lights, whose spectral output [20] coincides with the absorption spectrum of rose bengal. The insects, in cylindrical, screen-mesh
tubes, were surrounded by a vertical array of six fluorescent lamps. The
dimensions of each exposure tube were 34.0 cm in height and 8.9 cm in
diameter. One such exposure tube was constructed for each of four treatment
Photodynamically-Induced Clutathione Depletion
groups: DT,* DC, LC, and LT. The construction of the tube for LT contained
a clear glass funnel taped to the bottom to facilitate collection of moribund
insects. The tubes containing LC and LT were placed in the center of the
array of fluorescent lights. The dark treatment groups were kept in the dark
in the same room in tubes similar to those used for LC.
Four experimental groups were designated as noted above. A population
of insects was collected and separated into two treatment groups. One, a
control, was fed 10% sucrose in deionized water. The second group was fed
1mM rose bengal in 10% sucrose in deionized water. Both were allowed to
feed undisturbed in the dark for 24 h. At the end of this feeding time, each
of the two populations was divided into two subpopulations to yield the four
treatment groups. The DC and DT were kept in the dark for the same time
period for which the LT and LC were maintained under the lights. The LT
flies that dropped into the funnel during the first half hour were discarded.
As the moribund LT flies dropped into the funnel (during this time, only
rarely did any mortality occur in DT, DC, or LC), they were collected by
removing a cotton plug in the stem of the funnel, allowing the insects to fall
into liquid N2. The time of light exposure varied between 90 and 120 min,
the time required for a sufficiently large sample of moribund flies to be
collected. At the end of this time, LC, DC, and DT flies were collected by
immersion of their respective tubes in a canister of liquid N2.
The insects were stored at liquid N2 temperature until samples were
weighed for homogenization. Whole flies from each treatment group were
ground with a mortar and pestle precooled with liquid N2. Samples were
weighed into glass tissue grinder tubes. Extraction in the appropriate medium was by motor-driven grinding with a ground glass pestle.
Rose bengal (88% pure color) was obtained as the sodium salt (HiltonDavis Chemical Company, Cincinnati, OH). All other chemicals were of
reagent grade or better and were purchased from standard sources. All
absorbance measurements were made using a Gilford 2600 spectrophotometer. Fluorescence measurements were carried out on a Perkin-Elmer W F 44B fluorescence spectrophotometer.
Statistical treatment of the data was by analysis of variance for a split-plot
experimental design. Two treatments (control or dye-fed) were used, with
two factors (light or dark) within each; the means from each experiment were
used as replicates within each of the four resulting treatment groups. Probability values (p) were calculated for variation within treatments, factors (light
and dark), and replicates, as well as for the interaction between treatments
and factors, using the Statistical Analysis System (SAS Institute, Inc., Cary,
NC) on a Data General R/Iv/6000 computer. LSDs were also calculated, and
probabilities were obtained for differences between the means.
*Abbreviations: DC = nonlight-exposed, nondye-treated controls; DT = nonlight-exposed,
dye-treated flies; DTNB = 5,5'-dithiobis (Znitrobenzoic acid); HPLC = high-performance
liquid chrornatrography; LC = light-exposed, nondye-treated controls; LSD = least significant difference; LT = light-exposed, dye-treated flies; mBBr = rnonobromobimane; MTT =
3-(4,5-dirnethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; PES = phenazine ethosulfate;
TBA = thiobarbituric acid.
Wages and Heitz
Three a priori comparisons were made, on the basis of possible competing
induction and depletion, using these LSD values. These comparisons were
suggested by consideration that the final four treatment groups were treated
separately at two levels, those of dye-feeding for 24 h and those of light
exposure for approximately 2 h. This allowed more opportunity for ambient
conditions to increase experimental variation between, for example, LC and
LT than between DT and LT. Any difference between LC and LT may have
been generated over a time course of about 26 h; DT and LT have diverged,
however, over only 2 h. Any difference between DT and LT is thus potentially more important as a consequence of photodynamic action. Similar
considerations prompted comparisons between DC and LC to see the effect
of light exposure alone, and between DC and DT to see the effect of dyefeeding alone, as well as between DT and LT to observe the effects accompanying photodynamic action.
NADH and NADPH were extracted by homogenization in boiling 0.1 N
NaOH; NADf and NADPf were extracted in boiling 0.1 N HCl. The resulting extracts were then assayed by the continuous enzyme cycling microassay
of Matsumura and Miyachi [21].
Percent recoveries of NADPf and NADH added to acidic or basic extracts,
respectively, of both dye-fed and control insects, were reproducibly near
100%. This demonstrated that neither rose bengal nor an endogenous inhibitor was able to affect the assay.
For assay of total glutathione and thiol, extraction was by homogenization
in 20 mM EDTA. The extracts were kept at 100°C. for 3 min to denature
proteins. Centrifugation at 25,0009 for 5 min was sufficient to sediment
precipitated material. Total thiols were estimated using Ellman’s reagent [22].
Total glutathione was measured in the same extract using the cycling assay
of Owens and Belcher [23].
GSH, in abdomens from which the chitinous exoskeleton had been removed, was measured, using mBBr derivatization and HPLC separation as
described by Fahey and colleagues [24,25]. Standards were prepared by
derivatization of GSH, GSSG, Cys, cysteic acid, and CoASH, as described
by Fahey et al. [25].
HPLC separations were carried out using two chromatography pumps
from Waters Associates (Milford, MA) equipped with an automatic injector
(WISP 710A, Waters), a Perkin-Elmer LS-4 fluorescence spectrometer as detector, a data module (Waters Assoc.), and a programmable system controller
(Waters Assoc.). An Anspec Ultrasphere ODS (5 pm, C-18) column was
employed at ambient temperature and a flow rate of 1.5 mllmin, utilizing the
buffer system described by Newton et al. [26].
The GSH, Cys, and CoASH derivatives eluted at 11.0 f 0.5 min, 7.0 k 0.5
min, and approximately 30 min, respectively. Cys peaks were typically much
smaller than those for GSH, and many were not accurately quantifiable.
These data were omitted from analyses. CoASH peaks were barely detectable. No attempt was made to quantitate CoASH.
Experiments were designed to ascertain whether induction of total glutathione occurred in LC and DT. In the experiment to measure total glutathione
induction in LC, unsexed flies were fed ad libitum on 10% sucrose for 24 h
Photodynamically-Induced Glutathione Depletion
in the dark. Samples were collected as a function of illumination time.
Extracts were assayed for thiol and total glutathione as described above. In
the experiment designed to measure GSH induction in DT, two populations
of insects were fed ad libitum on either 1mM rose bengal in 10% sucrose or
10% sucrose alone in the dark. Samples were collected as a function of
feeding time and were assayed for GSH by derivatization with mBBr and
HPLC analysis as previously described.
S-n-Butyl homocysteine sulfoximine (buthionine sulfoximine), an inhibitor
of glutathione synthesis [27], was employed to deplete glutathione in a
population of insects to determine if glutathione depletion is a factor in
inducing mortality or in enhancing the phototoxicity of rose bengal. One
population was fed ad libitum on 10 mM buthionine sulfoximine in 10%
sucrose for 24 h in the dark; a second group was fed on 10% sucrose alone
under the same conditions. Total glutathione was estimated in these two
treatment groups as previously described. Samples of the remaining flies
were separated into clear plastic cups covered with screen wire. Each cup
contained 20 flies, with five cups per treatment. Four treatments were designated as follows: flies fed 10% sucrose for 24 h, then 10% sucrose for a second
24 h; flies fed 10% sucrose for 24 h, then 50 pM rose bengal in 10% sucrose
for 24 h; flies fed 1 O m M buthionine sulfoximine for 24 h, then 10% sucrose
for 24 h; and flies fed 10 mM buthionine sulfoximine for 24 h, then 50 pM
rose bengal in 10% sucrose for 24 h. At the end of the feeding period, all the
cups were placed under horizontal arrays of the fluorescent lights previously
described, and mortality was determined as a function of illumination time.
Mortality was compared between treatment groups to ascertain whether any
enhancement of toxicity by total glutathione depletion had occurred.
To measure lipid peroxidative potential, the TBA method adapted by Sohal
et al. [28] to measure lipid peroxidation in insect lipid extracts was used.
Recently, a fluorescence method [29] was described for direct determination
of hydroperoxides. This method was used to determine whether photodynamic treatment produced peroxidation products not readily detectable by
the TBA method. Extraction in ice-cold methanol, homogenization, and
reaction with dichlorofluorescin were carried out under an N2 atmosphere in
a polyethylene glove bag. Hematin dichlorofluorescin reagent was prepared
as described by Cathcart et al. [29]. For assay, 2.9 ml of the reagent was
mixed with 200 pl of the extract in a glass test tube, which was sealed and
incubated at 50°C. for 50 min. Fluorescence of dichlorofluorescein was read
at an emission wavelength of 523 nm and an excitation wavelength of 503
nm. The relative fluorescence in each reaction mixture was divided by the
wet weight of the tissue sample to obtain relative fluorescence per gram. This
value was employed in statistical analyses for comparison without extrapolation to actual lipid hydroperoxide concentrations.
Data from NADP+ and NADPH assays are given in Table 1.No significant
difference in NAD+ or NADH levels was observed (data not shown). Significance (p < 0.05) is found only in the difference between DT and LT with
Wages and Heitz
TABLE 1. Effect of Rose Bengal-Photosensitized Oxidation on NADP+ and NADPH Levels
in the Houseflv*
Dark (DC)
Light (LC)
Dark (DT)
Light (LT)
Dark (DC)
Light (LC)
Dark (DT)
Light (LT)
Coenzyme levels
(nmollg wet weight;
mean f SD)
Statistical comparisons
12.4 f 2.1
11.5 f 1.5
10.8 f 0.7
12.1 f 1.6
15.4 f 2.1
14.9 rt 1.4
16.1 f 2.6
13.4 zt 1.2
*N = five separate experiments. One homogenate was prepared per treatment in each of five
experiments on 5 different days. Each homogenate was assayed once.
TABLE 2. Effect of Rose Bengal-Photosensitized Oxidation on Total Glutathione (GSH +
GSSG) and Total Nonprotein Thiol Levels in the Housefly*
Total glutathione
Dark (DC)
Light (LC)
Dark (DT)
Light (LT)
Total thiol
Dark (DC)
Light (LC)
Dark (DT)
Light (LT)
Glutathione and thiol levels
(nmollg wet weight;
mean f SE)
Statistical comparisons
1,290 f 14
1,350 f 31
1,380 47
1,190 f 42
998 f 137
1,030 f 148
1,060 f 138
858 f 143
*N = Three separate experiments. Three homogenates per treatment were prepared in each
of three experiments on 3 different days. Each homogenate was assayed in triplicate.
respect to NADPH levels. The mean reduction in NADPH levels from DT to
LT is 16.7%. No concomitant, significant increase in NADP+ levels is observed, although it should be noted that the mean level in NADP' in LT is
11.6%greater (p < 0.15) than that in DT.
Total glutathione and total thiol were assayed (Table 2). Discrepancy between these values for total glutathione in the housefly and those obtained
by Saleh et al. [30] is probably due to the different extraction and assay
Photodynamically-Induced Glutathione Depletion
methods. Comparisons between treatment groups show significant differences in total glutathione between DC and LC, between DC and DT, and
between DT and LT and in total nonprotein thiol between DC and DT, and
between DT and LT.
Mean levels of GSH and Cys as determined by mBBr derivatization are
listed in Table 3. GSH titers, determined by this method, are lower than
those determined by the enzyme cycling method. Since care was taken to
maintain samples at liquid N2 temperature and to derivatize immediately, it
is doubtful that this discrepancy can be explained by oxidation of GSH to
GSSG. Another possible explanation is that extraction was less complete in
mBBr reagent than in boiling EDTA. A significant difference in GSH was
found between DT and LT. Differences in Cys content between DC and LC,
and between DC and DT, were also measured. The magnitude of depletion
of GSH in the LT from the levels in DT was approximately 55.7%.
Exposure of DC to light for varying time periods resulted in a general,
though small, increase (- 4.7%) in both total glutathione and thiol over time
(data not shown). The results from the treatment with rose bengal in the
dark indicated a generally higher total glutathione level ( 5-50% higher) in
DT than in DC (data not given).
A reduction of total glutathione to 19.5% of the control value was effected
by feeding 10 mM buthionine sulfoximine in the dark for 24 h. No effect on
mortality from total glutathione depletion was observed. No statistically
significant effects were found from photodynamic treatment by either the
TBA assay or the dichlorofluoresceinassay (data not given).
TABLE 3. Effect of Rose Bengal-Photosensitized Oxidation on Reduced Glutathione and
Cysteine Levels in the Housefly*
Dark (DC)
Light (LC)
Dark (DT)
Light (LT)
Dark (DC)
Light (LC)
Dark (DT)
Light (LT)
Levels of GSH and Cys
(nmolig wet weight;
mean f SE)
Statistical comparisons
825 & 11
711 f 130
973 f 309
431 f 103
210 f 32
105 +_ 21
110 k 16
94 f 18
*N = Three experiments. At least three homogenates were prepared per treatment. Each
homogenate was assayed at least two times, in each of three experiments on 3 different days,
Replication of cysteine assays was variable; data consisting of HPLC peaks that could not be
accurately measured were discarded.
Wages and Heitz
Lipid peroxidation has been observed to be associated with GSH depletion
[31,32]. Failure to observe an effect on lipid peroxidation by the TBA method
implies that photodynamic action leaves the oxidation state of housefly lipids
relatively untouched. That no effect is observed by either the TBA assay or
the dichlorofluorescein method suggests that photodynamic mortality in the
housefly does not involve lipid hydroperoxidation, which is well documented as an effect of in vitro xanthene dye-photosensitized oxidation. The
mass of literature on photooxidation of unsaturated fatty acids and cholesterol makes it apparent that some hydroperoxidation of membrane lipids
probably occurs. Failure to detect it in these experiments could be due to one
of the following: 1)Metabolism of lipid hydroperoxides rapidly follows their
formation such that only a negligible fraction remains until assay. 2) The
methods are insufficiently sensitive to detect hydroperoxidation of some
crucial lipid components, which represent only a small fraction of the total
lipid. 3) The insects expired from some other lesion before initial antioxidation barriers could be breached and detectable lipid hydroperoxidation could
occur. Yu et al. [33] showed that photooxidation of histidine residues led to
inactivation of membrane Ca2+ transport proteins before detectable lipid
hydroperoxidation occurred.
A significant GSH depletion was observed as a result of in vivo photodynamic action. The pool of total glutathione was also depleted, implying either
that GSH synthesis was inhibited or that GSH itself was oxidized to a
product other than the disulfide. NADPH depletion in the photodynamically
treated insects suggested oxidation of some amount of GSH to GSSG.
Assuming a relation between depletion of NADPH and GSH, at least some
of the GSH is being oxidized to GSSG, since the major enzyme involved in
maintaining the equilibrium between GSH and GSSG, GSSG reductase,
utilized NADPH as a donor of electrons for the reduction of GSSG. Irreversible conjugation of GSH with electrophilic products of photooxidation could
bring about the observed decrease in total glutathione. As a complete hypothesis, however, this is not consistent with the NADPH data. Lysis of
midgut epithelial cells [18] would bring intracellular GSH and the dye into
proximity in the lumen of the alimentary canal. Here, GSH would be expected to be photooxidized at a rate similar to that observed in vitro. Potentially, two separate mechanisms are operating under photodynamic
conditions: an oxidation of GSH to GSSG by oxidative detoxification mechanisms, which explains the depletion in NADPH levels, and an oxidation of
GSH to a product other than GSSG, possibly to the sulfonic acid or to an
irreversible conjugate.
A minor induction of total glutathione occurred within 2 h of light exposure. Given the general radioprotectant role of GSH, this is not surprising.
Induction of GSH also occurred with dye-feeding, which may be related to
the known role of GSH in detoxification of xenobiotics.
That a general GSH deficiency cannot account for observed mortality data
is shown by the inability of buthionine sulfoximine-mediated depletion of
total glutathione either to kill the insects or to synergize dye-sensitized
Photodynamically-InducedGlutathione Depletion
killing. The possibility remains that GSH depletion is occurring in response
to photooxidation of vital systems. In this case, depletion that would seem
insignificant in the context of normal experimental variation might, in fact,
be fatal. Unless localized depletion occurs, it appears that GSH depletion is
incident to, rather than the cause of, photodynamic toxicity.
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bengal, housefly, associates, musca, glutathione, photosensitizer, domestic, depletion, rose, mortality
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