Porphyric Insecticides - Chlorophyll Biosynthesis and Technological Applications - page 416

spectrofluorometry (Rebeiz 2002 ). Concomitant photodynamic damage was
assessed by monitoring the decrease in oxygen consumption of the incubated tissue
in the light. Oxygen consumption was determined polarographically, using a Clark
oxygen electrode.
The results of isolated organ and tissue investigations for each of the
aforementioned four insect species are summarized Table 18.10 .In T . ni , and
H. zea, significant Proto accumulation was observed in the midgut, and fat bodies.
Proto accumulation occurred when tissues were incubated with Dpy, ALA + Dpy,
Oph, and ALA + Oph (Table 18.10 ). No response to treatment with ALA alone was
observed. In cockroaches more of the Proto appeared to accumulate in the male and
female guts than in their abdomen. As in T . ni and H. zea, the response was elicited
by each of the treatments that included Dpy or Oph. Cotton boll weevil abdomens
appeared to be less responsive than the abdomens of the other three species.
To determine whether Proto accumulation resulted in photodynamic damage in
incubated tissues, T . ni midguts were incubated in darkness either in buffer, with
ALA, or with Oph + ALA. Oxygen consumption of the tissue was then monitored
before and after exposure to 2 h of illumination. It was assumed that decrease in O 2
consumption indicated photodynamic damage followed by cell death. A 30 %
decrease in O 2 consumption was observed in mid guts treated with Oph or with
ALA + Oph after 2 h in the light (Lee and Rebeiz 1995 ).
18.4.3 Subcellular Localization of Proto
Accumulation in T. ni
The decrease in oxygen consumption observed in isolated T . ni midguts
(Table 18.10 ), suggested that toxicity of porphyric insecticides may result, among
other things, from photodynamic damage to mitochondria. This issue was
investigated by Lee and Rebeiz as described below (Lee and Rebeiz 1995 ).
Fifth-instar T . ni larvae were placed on diets containing ALA (4 mM) and Oph
(3 mM) in darkness for 17 h. After dark-incubation, the site of Proto accumulation
in various subcellular components of the larvae was determined. Most of the Proto
was found in the mitochondrial (37 %) and microsomal (35 %) fractions, while the
balance (28 %) was found in the cytosol. In order to ascertain that the mitochondrial
Proto was not due to contamination by microsomal and cytosolic Proto, The Proto
content of mitochondria purified on Percoll gradients was also determined. Percoll-
purified mitochondria were highly active as evidenced by their succinate
cytochrome c reductase activity, and contained 534 nmol Proto per 100 mg mito-
chondrial protein. These results suggested that Proto formation may take place in
the mitochondria and microtomes both of which need Proto for heme formation,
while the presence of Proto in the cytosol may be due to leakage from the mitocho-
ndrial and microsomal compartments.