Diabetes was confirmed in streptozotocin (STZ)-injected rats by monitoring weight loss and a significant increase in blood glucose levels. Compared to control animals, plasma triglyceride and ALT levels were increased by factors of three and four, respectively, in the diabetic animals, while AST remained unchanged (data not shown). These parameters indicate that the diabetic animals had non-alcoholic fatty liver damage, which is consistent with the STZ model [29
]. One month after the onset of the disease, we used in vivo
spin-trapping techniques with EPR to assess free radical production in the bile of the animals. In spin trapping, short-lived free radical intermediates react with the spin-trapping agent, producing stable free radical adducts that are excreted in the bile. These can then be detected and characterized through their unique signature EPR spectra. Moreover, the fraction of the radicals trapped is proportional to the total amount of the given free radical produced, thereby allowing comparative quantitation [34
Experiments performed in diabetic rats led to the detection of strong six-line EPR signals of a POBN radical adduct which were reproducibly observed in the bile of rats 2 h after spin trap administration (). In age- and weight-matched controls, residual signals of POBN radical adducts were recorded (), confirming increased free radical formation in the diabetic animals. The free radicals trapped were identified through their EPR parameters, aN
= 15.75 ± 0.06 G and aβH
= 2.77 ± 0.07 G, corresponding to those reported previously for the POBN radical adduct of a carbon-centered, lipid-derived radical [32
Fig. 1 Free radical production in STZ-induced diabetes one month after the onset of disease. Representative EPR spectra of POBN radical adducts detected in bile of control (A) or diabetic (B) rats 2 h after POBN injection or in diabetic rats (C) 30 min after (more ...)
One possible candidate for triggering lipid peroxidation is the hydroxyl radical (•
OH). As the •
OH radical adduct of POBN is unstable, a •
OH scavenger, dimethyl sulfoxide (DMSO), was used to determine whether •
OH radicals were produced. It is well-known that •
OH is specifically converted into a methyl radical (•
) through its diffusion-limited reaction with DMSO (k = 7 × 109
]. The •
radical and closely related species are then trapped and detected by EPR as POBN-radical adducts. In rat bile, these POBN radical adduct signals increased significantly as early as 30 min after the simultaneous administration of POBN and DMSO in the STZ-treated rats (). Ex vivo
studies confirmed the in vivo
origin of the radical adduct formation. Only a minor background POBN radical adduct signal was noted following addition of POBN (20 mM) and DMSO (10 mM) in collecting tubes containing bile from diabetic rats (). The same characteristic POBN-lipid radical adduct was detected in the lipid extracts of kidneys from diabetic animals, but the intensity was only twofold higher than controls ().
Use of 13
C-labeled DMSO led to the detection of a twelve-line signal in diabetic, but not control bile (), providing further evidence for the fundamental role of the •
OH radicals in the diabetic animals. Computer simulation [38
] of the EPR spectrum () confirmed the presence of the POBN/•13
C-methyl, and possibly closely related species such as POBN/•13
OH (40) (aN
= 16.02 ± 0.02 G, aβH
= 2.98 ± 0.14 G, and a13
C = 4.59 ± 0.13 G). POBN/•
L was indicated by aN
=15.72 ± 0.11 G, aβH
= 2.92 ± 0.11 G [32
Fig. 2 Representative EPR spectra of rat bile obtained 30 min after injection of 13C-labeled DMSO (1 ml/kg) and POBN (1 g/kg). Spectrum of untreated control bile (A) showed no extra hyperfine splitting while diabetic bile (B) showed a twelve-line spectrum characteristic (more ...)
We next investigated in deeper detail the mechanism of free radical production and the possible origin of the •
OH produced in the progression of STZ-induced diabetes. For this, injections of known metabolic enzyme inhibitors in the diabetic rats were performed before the administration of the spin trap (). The xanthine oxidase inhibitor allopurinol (100 mg/kg, i.p.) [35
] given to rats 24 and 5 h before spin trapping with POBN had no effect on free radical generation in diabetic animals. Likewise, pretreatment with 1-aminobenzotriazle (ABT) (100 mg/kg, i.p.) [33
], a suicide substrate of cytochrome P450s, did not not affect the radical generation compared to the untreated diabetic group.
Fig. 3 Effect of different enzymatic inhibitors on free radical production in diabetic animals. (A) Comparative quantitation of the effects of different inhibitors and treatments on the detectable POBN radical adduct levels detected in diabetic rat bile one (more ...)
To study the possible role of transition metal ions in the generation of •
OH, diabetic animals were pretreated with the potent iron chelator Desferal (50 or 200 mg/kg, i.p., 1 h before spin trapping) [34
]. We also investigated whether phagocytic activation might be involved in, or related to, free radical production using gadolinium chloride (GdCl3
), a phagocytic suppressing agent (10 mg/kg, i.v.), 24 h before the experiments [36
]. Neither the iron chelator Desferal nor GdCl3
was found to cause any significant change in radical production in this disease model ().
In contrast, a dramatic decrease in the formation of free radicals was found in the diabetic animals when the non-specific nitric oxide synthase (NOS) inhibitor aminoguanidine (200 mg/kg, i.p.) was administered 30 min prior to the POBN/DMSO injection [37
] (). This effect was even more pronounced when rats were injected with 1400W (15 mg/kg, i.p.), a specific inhibitor of the inducible form of NOS (iNOS) [41
], 1 h before spin trap administration (). The inhibiting effect remained for over 2 h (data not shown), and only a residual EPR signal of POBN radical adducts was detected 30 min after injection with POBN/DMSO. To determine if 1400W affected the yield of the lipid and the methyl radicals trapped in the presence of DMSO differently, we performed additional studies with 13
C-DMSO and 1400W. As shown in , the hyperfine structure of the spectrum was not changed from , thus indicating that both methyl and lipid radicals were secondary species derived from the •
OH reaction with DMSO and lipids, respectively.
The lack of an effect from Desferal revealed that hydroxyl radical production was independent of iron catalysis, suggesting peroxynitrite production since spontaneous peroxynitrite decomposition is a known metal-independent source of hydroxyl radicals. In addition, none of the metabolic inhibitors tested other than the iNOS inhibitors inhibited the free radical formation. This suggests a close relationship between nitric oxide and subsequent •
OH production. Taken together, these results suggest a significant contribution of iNOS in the mechanism. Nitric oxide synthase have also been demonstrated to be able to produce both •
NO and O2•−
] and could thus be the source of peroxynitrite and, consequently, •
OH. Other possible sources of superoxide, such as the mitochondria, and NADPH oxidases cannot be excluded as well in our model but we have no data to confirm that. To further test the role of iNOS, diabetic rats were injected with l
–arginine (100 mg/kg) 30 min before applying POBN. This treatment should diminish O2•−
generation that occurs in the absence of l
-arginine and, thus, peroxynitrite production. Indeed, l
–arginine pretreatment consistently prevented lipid radical formation, leaving only a minor background EPR signal of the POBN adduct ().
Fig. 4 Effect of l-arginine pretreatment on free radical generation in diabetic animals. (A) l-arginine administration (100 mg/kg) reduced the detectable free radical level to the background signal. Spectra show bile from an untreated diabetic rat and from a (more ...)
In addition to these mechanistic studies, time course EPR experiments of bile collected from animals 1, 2 and 3 weeks following STZ injection revealed significantly increased lipid radical production after three weeks of diabetes compared to control animals (). Together with increased formation of free radical intermediates at 3 weeks, iNOS protein expression was considerably augmented in the liver and in the kidney, known target organs for diabetes (). In tissues that are not affected in diabetes such as the lung, iNOS overexpression was not observed ().
Fig. 5 Time course study of lipid radical adduct production measured by EPR spectroscopy. POBN/lipid radical adducts were determined in the bile of diabetic and age-matched control rats one, two, three and four weeks after the onset of the disease. Each experiment (more ...)
Fig. 6 Western blot analysis of iNOS protein expression in rat liver (A), kidney (B) and lung (C) tissues after one month of diabetes. Data are representative of three independent experiments. Graph shows the statistics between control (white bar) and diabetic (more ...)
To gather further insight about iNOS expression patterns in the liver of diabetic animals and to further examine the key role of peroxynitrite in the concomitant protein damage, confocal microscopy of in situ fixed liver slices obtained from control and diabetic animals was performed. Nitrotyrosine was utilized as a marker of protein modification/oxidation resulting from increased reactive species production. As shown in ., increased iNOS immunostaining was apparent in the diabetic liver and was consistently more intense around vessels of pericentral hepatocytes (). Positive nitrotyrosine staining was also observed in the diabetic slices (), and presented considerable colocalization with iNOS (). This data suggests that iNOS overexpression led to protein nitration, which is consistent with peroxynitrite formation.
Fig. 7 Immunofluorescence detection and colocalization of iNOS and nitrotyrosine in rat liver tissues using confocal microscopy. (A) Significant iNOS staining was observed in diabetic rat liver (green labeling). (B) Liver slices from control animals show little (more ...)
Additional immunohistochemical studies revealed significantly increased 4-hydroxynonenal formation and conjugation to proteins in the liver () and kidney () of diabetic animals. 4-Hydroxynonenal is a well characterized aldehyde product of lipid peroxidation which reacts with protein amine groups to produce chemical modification of proteins and enzymes, thus possibly contributing to the complications of diabetes (). In the lung, which is not a target organ, low levels of lipid peroxidation were detected in comparison with controls, which was consistent with the absence of iNOS overexpression in that organ (). Taken together, our results strongly support a mechanism where EPR-detectable radical adducts in diabetic bile and kidney are iNOS-dependent, possibly leading to increased lipid peroxidation and protein modification.
Fig. 8 Immunohistochemical detection of 4-hydroxynonenal in the diabetic liver, kidney and lung. (A) Control liver samples showed minimal staining; meanwhile, (B) strong positive staining was observed in the diabetic liver. (C) 4-hydroxynonenal detection in (more ...)