Influence of Animal Species on Secondary Oxidant Activation in Serum Exposed to Singlet Oxygen

Isabelle Lhommeau, Samuel Douillard, Antoine Foursac, Lorena Aillet, Edith Bigot, and Thierry Patrice

Isabelle Lhommeau  and Antoine Foursac are technicians, Samuel Douillard is an assisting engineer, Lorena Aillet is a pharmacist, all belonging to the LaserDepartment/Cancer Photobiology Laboratory. Thierry Patrice, MD, PhD, is the head of the LaserDepartment /Cancer Photobiology Laboratory, and Edith Bigot, PhD, is a laboratory biochemist at the Laënnec Hospital, CHU de Nantes, 44093, Nantes, France.

Address correspondence and reprint requests to Professor T. Patrice, Laboratoire de Photobiologie des Cancers, Département Laser, 44093 Nantes, France, Tel: (33) 2-40-16-56-75 or (33) 2-40-16-53-37, or email: thierry.patrice@chu-nantes.fr.

¹Abbreviations that appear ≥3× throughout this article:  AUC, area under the curve; DCF, 2ˈ-7ˈdichloro-fluorescein (oxidized, fluorescent); DCFH, activated 2ˈ-7ˈ dichloro-dihydro-fluorescein (reduced); FCS, fetal calf serum; Hb, hemoglobin; HS, human serum; NADPH, nicotinamide adenine dinucleotide phosphate; O₂,singlet oxygen; OD, optical density; PDT, photodynamic therapy; RB, rose bengal; RBC, red blood cells; ROS, reactive oxygen species; SD, standard deviation; SOS, secondary reactive oxygen species and peroxides.

Abstract

Singlet oxygen (¹O₂) produced during inflammatory reactions and during photodynamic therapy deactivates by producing in tissues secondary reactive oxygen species and peroxides (SOS) as well as other degradation products. We investigated the influence of animal species on SOS production secondary to standardized ¹O₂ production by performing in vitro experiments with rose bengal as the ¹O₂ producer, human serum (HS) as a control, sera derived from various animal species, and dichloro-dihydro-fluorescein as a nonspecific marker that becomes fluorescent when oxidized. The overall SOS production in HS from a presumed healthy cohort of 53 donors (31 males and 22 females) gave a mean “normal” value of 0.91 compared with a previous pool of 75 male sera samples. SOS production after a photo-reaction was two or four times lower in HS than in fetal calf serum or mice sera, respectively. In mice, the “nude” characteristic increased even more than in the SOS production. In the Aves order, this production appeared to be distributed randomly according to the number of branches after the appearance of Amniotas. For primates, SOS production appeared to decrease linearly with the number of branches (R² = 0.98). Adding hemolysates from complete bloods to the corresponding sera induced an increase in SOS production in all species, proportional to the production in sera. These findings should be kept in mind when interpreting results from studies of secondary reactive oxygen species-induced pathways following ¹O₂ production, regardless of its origin.

Key Words:  dichlorofluorescein; hemolysis; oxidative stress; primates; reactive oxygen species (ROS); serum; singlet oxygen

Introduction

Photodynamic therapy (PDT¹), a procedure used during the course of human and, more recently, veterinary practice (Ferreira and Rahal et al. 2009; Marconato and Buchholz et al. 2012) is based on the production of singlet oxygen (¹O₂¹)(Weishaupt and Gomer et al. 1976) and reactive oxygen species (ROS¹) (P. V. Nariharan and J. Courtney et al. 1980; Ochsner 1997) under light exposure, leading to tissue death. During PDT photosensitizers may reach some specific targets and can be administered through various routes or vehicles, including intravenous injections of different durations or can be applied topically. Light emitted at a wavelength absorbed by the photosensitizer can also be delivered at various intervals following sensitizer administration and according to a range of parameters. Such oxidative species are also deactivated through the production of even more oxidizing species that randomly induce damage to various biological targets (Tanielian and Mechin et al. 2000; Kon and Tanigawa et al. 2004). ¹O₂, an excited form of “normal” molecular O_2, and ROS are also involved as a cause or a consequence of many diseases, inflammatory reactions, and metabolic pathways. We recently demonstrated that secondary ROS and peroxides production was much lower in human serum than in fetal calf serum (FCS¹), with production differing from one person to another (Olivier and Douillard et al. 2009b). The aim of the present study was to determine whether sera obtained from the blood of several animal species had the same capacity for secondary ROS and peroxides (SOS¹) neutralization as sera prepared from human blood. If this is not the case, it would mean that neutralization of SOS does not occur similarly, as neutralization would take place physiologically through the consumption of normally present antioxidants. To address this question, we delivered a standardized amount of photo-produced ¹O₂ to sera and measured the induced SOS_.

In the present report we assess the influence of the animal species origin, with a particular focus on primates, of serum on SOS production and/or deactivation. We also discuss the influence of hemolysis of respective types of blood on this production.

Materials and Methods

Chemicals

We diluted rose bengal (RB¹, CAS [632-69-9], from Sigma-Aldrich, France), a photosensitizer with a quantum yield of ¹O₂ production as high as 75% (Redmond and Gamlin 1999), in sterile water for injections.  We then prepared activated dichloro-dihydro-fluorescein (DCFH¹) from 2ˈ–7ˈ dichloro-dihydro-fluorescein diacetate (DCFH-DA, Sigma, Saint Quentin Fallavier, France) and diluted the DCFH before adding it to the samples to be assayed (1 mL, 40 µM) immediately after light delivery to monitor oxidative species (Hafer and Iwamoto et al. 2008).

Biological Reagents

Human Sera (HS)

We isolated a total of 53 human serum (HS¹) samples (from 22 women and 31 men) and assayed the sera individually for secondary ROS and peroxides production. We then compared the results with blood samples from a first cohort of healthy blood donors (from n = 75 men) recruited consecutively for whom sera had been pooled (EFS 1). The study was conducted according to the protocol (NTS 2006-02) established between the Etablissement Français du Sang and the Nantes University Hospital, according to the Helsinki declaration (1964/2000). Eating and smoking were forbidden during the 2 hours before blood sampling as described in the study exclusion criteria. We carefully drew a total of 10 mL of venous blood, avoiding hemolysis as much as possible by using Terumo^® Vacutainers and Terumo^® Venoject Quickfit needles on sterile clot act dry tubes (Terumo^® Venosafe VF-054SP). We allowed the blood to coagulate at room temperature for 30 minutes before it was centrifuged at 3500 rpm for 10 minutes. We then carefully collected the HS, avoiding any hemoglobin aspiration under sterile conditions, and separated the HS into two aliquots in 5-mL sterile tubes (Starlab^® France, Paris, ref TK75-006) that we immediately froze at -20°C until it was processed. The maximum time between blood sampling and freezing was less than 40 minutes.

Origin of Animal Sera

For comparison purposes, we used FCS in some experiments. We obtained the FCS that we used for cell culture from Bio Whittaker, Cambrex, Belgium (Aliquot N° 5SB002). Rat (Rattus norvegicus) serum was prepared from Sprague Dawley rats (Elevage Janvier, Le genest St Isle, France). Mouse (Mus musculus) serum was prepared from 10 RjOrl:SWISS mice, 10 RjHan:NMRI mice and 10 Rj:NMRI nude mice (Elevage Janvier, Le genest St Isle, France). The mice were anesthetized by inhalation of ether for 1 minute and blood was collected by intracardiac puncture (0.2 mL, 26 Gauge needle [Nipro^® Osaka, Japan]). Serum samples were pooled per species and frozen at -20°C until use. In the remainder of this text, murine species are named by their strain.

We obtained the following primate (excluding human) and pre-primate samples at the Strasbourg University Primatology Center: marmosets (Callithrix jacchus) and vervets (Chlorocebus aethiops); at the Centre National de la Recherche Scientifique Primatology Center in Marseille, baboons (Papio papio) and squirrel monkeys (Saïmiri sciureus); and at the UMR CNRS/MNHN 7179 (Dr. Aujard, Brunoy, France), mouse lemurs (Microcebus murinus). The following bird serum samples were provided by the Nalet farm (La Chapelle sur Erdre, France): chickens (Strain JA 957), guinea fowls (Numida meleagris), and laying hens (Gallus gallus domesticus).  Muscovy ducks (Cairina moschata) and ostriches (Struthio camelus) were provided by “La ferme aux autruches” (Guidel, France). Samples from crocodiles (Crocodylus niloticus) were provided by Dr. S. Martin, “La ferme aux crocodiles” (Pierrelatte, France). All animals from a given species had been fed and had grown up in a highly similar manner.

The number of branches within the tree of life had been calculated for all species from the tree of life project (Maddison and Schulz et al. 2010). The branches were counted from the appearance of both Amniotas and primates according to the latest possible discoveries, although a certain lack of precision could have been present in the Aves order. This number does not reflect discussions on the topic but rather the most commonly recognized shape of the tree of life.

Absorbance Measurements

We measured absorption spectra (380-800 nm) with a Techcomp^® 8500 UV/VIS absorption spectrophotometer (Fischer, France) for each patient sample and control following dilution in sterile water (final = 5% serum). The blank was the same sterile water placed in a similar cuvette.

Analysis of ¹O₂-Induced SOS Deactivation

The measurement principle (Olivier and Douillard et al. 2009b) that we used was based on the analysis of ROS and peroxides production, induced by photodynamically produced, by means of the DCFH/dichloro-dihydro-fluorescent (DCF¹) system after adding activated DCFH to each sample assayed immediately after the end of light delivery. Briefly, we prepared a solution extemporaneously in glass cuvettes specialized for fluorescence (1 cm x 1 cm). The solution consisted of serum and RB (initially at 100 µg/mL) dissolved in sterile water for injections (pH 7). We then performed the 514-nm laser (Ar-ion Inova 70, Coherent^®) irradiation in the cuvette containing 500 µL of solution. The laser beam was transmitted to the target by a silica-silicon step-index fiber (core diameter 600 µm) (Quartz et Silice^®, France) that provided a 10 mm-diameter laser spot and illuminated the solution. Immediately after irradiation, we added activated DCFH (1 mL, 40 µM). The DCFH solution oxidation resulted in the formation of DCF (excitation 488 nm, emission 525 nm). We began to record the fluorescence spectra of solutions containing sensitizer, serum, and DCF 40 seconds after the end of light delivery for 66 minutes with a monitored temperature of 40°C. For these experiments, we used a Varian^® Cary Eclipse spectrofluorimeter to enable the assessment of fluorescence levels for four cells simultaneously coupled to a Varian^® Cary PCB 150 Peltier device. We measured the area under the curve (AUC¹) of change in DCF fluorescence over time for each serum, and we calculated the raw ratio by dividing the value of the tested serum by that of the reference (EFS 1).

Influence of Hemolysis

We obtained a sample of complete blood (ethylene diamine tetraacetic acid K₂ tubes) in parallel for each serum sample, which we fully hemolyzed by ultrasound (Vibracell 72434, probe CV18/1524, power 40, three cycles of 1 minute per sample). We then mixed the sera with increasing concentrations of the corresponding complete blood samples and repeated the assay. We used complete blood dilutions (0, 1/250 to 1/2000) that corresponded to hemoglobin concentrations of 0.0967, 0.058, 0.029, 0.0193, 0.0145, 0.00967, 0.00725, and 0.0058 g/dL for HS assays. We then assayed each solution as described above for DCF fluorescence after RB addition (5 µg/mL) and light treatment (514 nm, 20 J/cm²), as well as for absorption spectrum measurement, after which we drew a graph to depict the evolution of DCF fluorescence according to optical densities (ODs¹).

Results

The capacity of SOS production varied from one healthy person to another (Olivier and Douillard et al. 2009b) but was constantly lower than in other primates. It had been found to be higher for Microcebus murinus (pre-primates), Platyrrhini, Catarrhini, and among them for humans (Figures 1 and 2), in this order of magnitude, thus following the phylogeny of primates (classified accordingly to the existence of primitive anthropomorphic characters (Thorington and Anderson 1984)). Pooling SOS production values according to the number of divisions of the primate order to reach a given species (Maddison and Schulz et al. 2010) confirmed that there was a direct relation between the increase in ROS and peroxides production and the number of branches within primates (R² = 0.98) (Figure 2). All series of SOS production ratios were normally distributed (Kolmogorov-Smirnov test) and, with the exception of Callithrix jacchus versus Chlorocebus aethiops, which were statistically different from each other (Mann-Whitney U test, p < 0.05).  When pooled according to the number of branches within primates, the series of ratios (assayed serum AUC/HS pooled sera AUC) correlated with the number of branches (Kruskal-Wallis one-way analysis of variance on ranks, p < 0.001, Dunn’s method, p < 0.05).

 

 

 

SOS in Mammals Other Than Primates

In a previous paper we (Olivier et al. 2009b) noted that the capacity for SOS production widely varied between FCS and HS. In the current study, using similar experimental conditions (RB and serum concentration, temperature, and irradiation) for each serum tested, our data indicate that the capacity of mouse sera for SOS production was about twice as high as that of FCS, and four times higher than that of HS. We found that secondary ROS production in rats was nearly three-fold higher than in HS, and that not all mice had a similar capacity for SOS production. In fact, the sera from Swiss and NMRI mice showed different patterns. Moreover, the “nude” characteristic induced changes in a given strain in that nude NMRI produced 14% more ROS than their non-nude counterparts (Table 1).

Table 1. Secondary reactive oxygen species or peroxides production ratio as a function of mean morphological standards^{a, b}

SOS in Birds and Crocodiles

As shown in Figure 3, representatives of the Aves class revealed different results, although the number of species studied was limited. We found ROS and peroxides production to be significantly higher for birds than for primates for a comparable number of branches, and the number of branches did not appear to influence SOS production significantly. Guinea fowls and chickens resulting from 24 +/-2 branches from the appearance of Amniotas had a ratio of 5.78 (standard deviation [SD¹] 1.39) and 4.14 (SD 1.36), respectively, whereas ducks, which are 19 +/- 2 branches away from appearance of the Amniotas, and Struthio camelus, which are 15 +/-2 branches away, also had a lower SOS production, at 4.78 (SD 1.71) and 4.64 (SD 1.51), respectively. In addition, we found SOS production to be better in crocodiles (ratio 3.79, SD 1.66) than in Struthio camelus, although the former species results from only 10 known divisions within the Amniotas compared with 15 for the latter.

 

 

Biochemistry and Influence of Hemolysis on SOS Production

Although the routine biochemical and hematological parameters were all within the normal published ranges, the capacity of hemolysates to deactivate serum ROS and peroxides production differed from one species to another, with the hemolysates of birds deactivating significantly less than those of primates. When adding pooled fully hemolyzed whole bloods, and reconstructing the AUC for the ROS and peroxides production according to OD  at 413 nm, a proportional increase in ROS and peroxides production was noted for each species studied (Figure 4). The shape of the curves differed considerably from one species to another but reflected the capacity of sera to produce SOS. For some species, such as the Microcebus murinus or Saïmiri sciureus, the production of SOS was low in the absence of added hemolysate but rapidly changed when the OD at 413 nm increased. However, in certain other species, such as N. meleagris or G. gallus domesticus, a huge amount of hemolysate had only a limited impact on SOS production.

 

 

We also analyzed potential correlations between biochemical parameters described in the literature as influencing secondary SOS production or oxidative stress. FCS and lemur sera showed a different level of creatinine and bilirubin, respectively, compared with the other species. C. niloticus and Aves had a lower rate of urea than mammals in which a trend toward a relation with SOS production was observed. Total cholesterol and glucose correlated roughly with capacity for SOS production, whereas an inverse correlation was observed for proteins (Figure 5). Finally, ultrasound hemolysis did not modify total iron, transferrin-bound iron, and ferritin concentration. They did not exert any influence on SOS production whatever the species studied.

 

Discussion

¹O₂ is an excited form of molecular oxygen that is strongly oxidative for many biological targets, either directly or indirectly through the successive formation of various oxidative species. It is produced in vivo by activated neutrophils (Badwey Karnovsky 1980) or eosinophils (Kanofsky and Hoogland et al. 1988) and during various biochemical reactions including energy production (Kanofsky 1989) and photoreactions (Weishaupt and Gomer et al. 1976). ¹O₂ is neutralized through antioxidative processes or after damaging biological tissues, and it may react with various targets and/or generate ROS that are, in some cases, nearly as powerful as ¹O₂  but with a much longer half-life (Kessel and Luo 1996; Wright and Hawkins et al. 2003; Girotti 2008). The production of ¹O₂ requires oxygen, but its overall effect also depends on the way it is deactivated into SOS. In addition, the very limited radius of action of ¹O₂, (Moan and Berg 1991; Snyder and Skovsen et al. 2006), due to its short lifespan, makes additional mechanistic pathways plausible to explain all described effects, increasing the sole ¹O₂ effects (Chakraborty and Held et al. 2009; Olivier and Douillard et al. 2009a).

In a previous paper we found the capacity of ¹O₂ deactivation to vary widely between FCS and HS (Olivier and Douillard et al. 2009b). In the current study, we used similar experimental conditions (RB and serum concentration, temperature, and irradiation) for each serum tested, and we extended the range of species studied to animals that are only rarely assessed for their resistance to ROS. We show that the capacity of mice sera for SOS production is about twice as high as that of FCS, and four times higher than that of HS. This potential has also been noted, although rarely addressed, in other species (Ninfali and Aluigi 1998). In addition, not all mice species had a similar capacity for production. For example, the sera from Swiss and NMRI mice showed different patterns. This difference, which is probably related to sera compositions that vary greatly from one species to another and accordingly influence ROS production, should lead to careful interpretation of ¹O₂ or ROS effects, whatever their origin. Moreover, in mice the nude characteristic induced changes in a given species; nude NMRI produced 14% more ROS than their non-nude counterparts. Although biochemical differences between strains are conceivable, the underlying reasons for such differences related to the “nude”characteristic of mice are difficult to understand. These results are challenging because they complicate the extrapolation of experimental data from one model to another and of course from animals to humans, particularly when assays involve oxidative treatments, which is often the case in oncology and always the case during PDT. In addition, differences in resistance to ROS have previously been related to the mean lifespan of species (Sohal and Sohal et al. 1990). However, SOS production did not appear to correlate with the life expectancy of the species included within this study (Table 1) or of nonprimates or primates (data from the literature, e.g., Sohal et al. 1990).

Our data are concordant with those of Ninfali (Ninfali Aluigi 1998), obtained using the oxygen radical absorbance capacity (“ORAC”) test to assess the total antioxidant capacity instead of resistance to ¹O₂ and ROS. They observed a three-fold lower protection against ROS in chickens compared with Caucasian humans, a value close to the 4.86-fold noted in our study and a 6.4-fold higher SOS production for N. Meleagris. Based only on this result, we were unable to explain the difference between chickens (Strain JA 957) and N. Meleagris, which are similar to each other from a cladistic point of view. In parallel we assessed the capacity of the hemolysates to reduce the SOS production of the corresponding serum for each species. Whereas the routine biochemical and hematological parameters were all within the normal published ranges, the capacity of hemolysates to deactivate serum SOS production differed from one species to another, and hemolysates of birds deactivated significantly more of the corresponding sera SOS production than those of primates. One could hypothesize that these differences are related to the presence of nuclei within avian red blood cells (RBCs¹), leading to a lower amount of antioxidative enzymes at the time of the analyses. When adding pooled fully hemolyzed whole bloods and reconstructing the AUC obtained for the ROS and peroxides production according to OD at 413 nm, an increase in SOS for each species studied (Figure 3) could be noted, roughly following the initial value of SOS production that corresponded to the lowest OD. The influence of hemolysates on ROS and peroxides production is in agreement with the observation by Sohal and colleagues (Sohal and Sohal et al. 1990) that glutathione peroxidase activity negatively correlates with life expectancy in mammals. Iron could have been released from hemoglobin during ultrasound treatment, which could have induced more SOS through the Fenton reaction. Serum transferring-bound iron had been shown to be only poorly influenced by sonication. In addition, in another series of experiments, attempts made to modify SOS production by adding iron to human sera had been unsuccessful (unpublished data).

RBCs are present in human blood at a concentration of 4.5 x 10⁶/mL and are specialized in carrying oxygen and protons (Cataldi Di Giulio 2009) by means of the specialized molecule hemoglobin (Hb¹). RBCs are loaded with antioxidant systems specialized to prevent Hb damage due to the oxidative activity resulting from O₂ transport or from other oxidants (Andersen and Nielsen et al. 1997; Azarov and Huang et al. 2005). Homeostasis of RBCs causes energy release through glycolysis, itself producing ROS via the nicotinamide adenine dinucleotide phosphate (NADPH¹)/NADPH₂ system (Kawahito and Kitahata et al. 2009). Because RBCs are non-nucleated in humans, they must retain the appropriate enzymatic content throughout their entire lifespan of about 120 days. The RBC membrane thus acts as a filter aimed at quantitatively protecting its content and avoiding its alteration as well as permitting the transfer of glucose, ions, and gases such as O₂. One could hypothesize that differences in the antioxidant content of RBCs could be related, to a certain extent, to the presence of nuclei within avian RBC, allowing a lower amount of antioxidative enzymes at the time of analysis.

However, RBC content appears to strongly increase serum ROS or peroxides production after ¹O₂ production, making it seemingly incompatible with serum as it decreases defenses against oxidative species. Several authors have reported that hemoglobin could scavenge nitrous oxide^. at such a high rate that it makes HB become oxidative (Nagababu Rifkind 2004). When the RBC membrane exists, it separates the RBC content from the main antioxidant circulating system(s) located in the serum without interfering with RBC metabolism. These systems could be made up of proteins that are present in such quantities that they can efficiently deactivate oxidative species potentially produced secondarily to primary ROS or ¹O₂ without themselves becoming toxic. Albumin is one of several serum proteins that have an antioxidative cystein 34 residue (Summa and Spiga et al. 2007). Insulin carries three di-sulfur bonds for 51 amino acids, which could partly explain its antioxidative role in addition to its glycemia-regulating function, although the ROS neutralization is less when sulfur is involved in a bond compared with a cystein residue (Bashan and Kovsan et al. 2009). Haptoglobin, in addition to its cystein residues, interacts with free Hb to decrease its oxidative effects (Wicher   Fries 2006; Boretti and Buehler et al. 2009; Levy and Asleh et al. 2010). Finally, one should consider that serum ROS and peroxides production are functions that were inherited during species evolution at roughly the same time as the function of oxygen in metabolism.

One could hypothesize that in the absence of a RBC membrane, the overall ROS and peroxides production should be less in hemolymph than in serum because the RBC content increases ROS after ¹O_2, and therefore the consequences of inflammatory or more deleterious metabolic reactions would have to be down-regulated to a lower and probably less efficient level (all other parameters being supposed identical). The species from the Aves class within our study shared the characteristic that none of them fly, which could explain the fact that the values were relatively similar (Moller 2007). The fact that birds do not follow the same pattern could be due to the limited number of species, selected abnormal patterns due to breeding, a too-imprecise cladistic tree, or even that a better evolution-dependent ROS and peroxides production system is specific to primates (Wicher and Fries 2006). It could be a determinant of primate evolution — a better ROS and peroxides production that provides a greater metabolic adaptability (Dufour and Landick et al. 2008). It is likely that a large number of genes influenced ROS and peroxides production during evolution of species; their identification could be the subject of further studies. Finally, it appears that SOS production in serum, which is not without effects, is largely species dependent, although the significance of this characteristic is unclear. Several biochemical parameters that have been measured have varied according to species and have eventually correlated with secondary SOS production. However, some of the characteristics that were likely to influence this production varied inversely (e.g., glucose [(Bourdon and Loreau et al. 1999; Schleicher and Friess 2007), creatinine (Seyama 1993), cholesterol (Kessel and Luo 1996)) so that the exact role of each remains impossible to predict at this step of our study. Urea concentration had been found to differ between mammals and birds studied because they excrete nitrogen as uric acid (Sheldon and Hoover et al. 2007).

Conclusion

One could hypothesize that secondary ROS production after primary ROS also reflects the capacity for ¹O₂ to deactivate. Because ROS or ¹O₂ production is a very ancient pattern that appeared in living species concomitantly with their exposure to oxygen and later to sun, one could hypothesize that deactivation pathways may also result from this long evolution, regardless of the source of ROS or ¹O₂  (Reischl and Dafre et al. 2007).

Acknowledgments

We are grateful to Joanna Ashton-Chess for help in manuscript translation and to Etablissement Français du sang for their support. This work received funding from the Ligue Nationale contre le Cancer.

References

Andersen HR, Nielsen JB, Nielsen F, Grandjean P. 1997. Antioxidative enzyme activities in human erythrocytes. Clin Chem 43:562-568.

Azarov I, Huang KT, Basu S, Gladwin MT, Hogg N, Kim-Shapiro DB. 2005. Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation. J Biol Chem 280:39024-39032.

Badwey JA, Karnovsky ML. 1980. Active oxygen species and the functions of phagocytic leukocytes. Annu Rev Biochem 49:695-726.

Bashan N, Kovsan J, Kachko I, Ovadia H, Rudich A. 2009. Positive and negative regulation of insulin signaling by reactive oxygen and nitrogen species. Physiol Rev 89:27-71.

Boretti FS, Buehler PW, D'Agnillo F, Kluge K, Glaus T, Butt OI, Jia Y, Goede J, Pereira CP, Maggiorini M, Schoedon G, Alayash AI, Schaer DJ. 2009. Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs. J Clin Invest 119:2271-2280.

Bourdon E, Loreau N, Blache D. 1999. Glucose and free radicals impair the antioxidant properties of serum albumin. FASEB J 13:233-244.

Cataldi A, Di Giulio C. 2009. "Oxygen supply" as modulator of aging processes: hypoxia and hyperoxia models for aging studies. Curr Aging Sci 2:95-102.

Chakraborty A, Held KD, Prise KM, Liber HL, Redmond RW. 2009. Bystander effects induced by diffusing mediators after photodynamic stress. Radiat Res 172:74-81.

Dufour YS, Landick R, Donohue TJ. 2008. Organization and evolution of the biological response to singlet oxygen stress. J Mol Biol 383:713-730.

Ferreira I, Rahal SC, Rocha NS, Gouveia AH, Correa TP, Carvalho YK, Bagnato VS. 2009. Hematoporphyrin-based photodynamic therapy for cutaneous squamous cell carcinoma in cats. Vet Dermatol 20:174-178.

Girotti AW. 2008. Translocation as a means of disseminating lipid hydroperoxide-induced oxidative damage and effector action. Free Radic Biol Med 44:956-968.

Hafer K, Iwamoto KS, Schiestl RH. 2008. Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations. Radiat Res 169:460-468.

Kanofsky JR. 1989. Singlet oxygen production by biological systems. Chem Biol Interact 70: 1-28.

Kanofsky JR, Hoogland H, Wever R, Weiss SJ. 1988. Singlet oxygen production by human eosinophils. J Biol Chem 263:9692-9696.

Kawahito S, Kitahata H, Oshita S. 2009. Problems associated with glucose toxicity: role of hyperglycemia-induced oxidative stress. World J Gastroenterol 15:4137-4142.

Kessel D, Luo Y. 1996. Delayed oxidative photodamage induced by photodynamic therapy. Photochem Photobiol 64:601-604.

Kon T, Tanigawa T, Hayamizu K, Shen M, Tsuji T, Naito Y and Yoshikawa T. 2004. Singlet oxygen quenching activity of human serum. Redox Rep 9: 325-330.

Levy AP, Asleh R, Blum S, Levy NS, Miller-Lotan R, Kalet-Litman S, Anbinder Y, Lache O, Nakhoul FM, Asaf R, Farbstein D, Pollak M, Soloveichik YZ, Strauss M, Alshiek J, Livshits A, Schwartz A, Awad H, Jad K, Goldenstein H. 2010. Haptoglobin: Basic and clinical aspects. Antioxid Redox Signal 12:293-304.

Maddison DR, Schulz KS, Maddison WP. (010. "Tree of life web project." Online (http://tolweb.org/tree/).

Marconato L, Buchholz J, Keller M, Bettini G, Valenti P, Kaser-Hotz B. 2012. Multimodal therapeutic approach and interdisciplinary challenge for the treatment of unresectable head and neck squamous cell carcinoma in six cats: A pilot study. Vet Comp Oncol (In Press). 2012 Mar 23. doi: 10.1111/j.1476-5829.2011.00304.x [Epub ahead of print].

Moan J, Berg K. 1991. The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen. Photochem Photobiol 53:549-553.

Moller AP. 2007. Senescence in relation to latitude and migration in birds. J Evol Biol 20: 750-757.

Nagababu E, Rifkind JM. 2004. Heme degradation by reactive oxygen species. Antioxid Redox Signal 6:967-978.

Ninfali P, Aluigi G. 1998. Variability of oxygen radical absorbance capacity (ORAC) in different animal species. Free Radic Res 29:399-408.

Ochsner M. 1997. Photophysical and photobiological processes in the photodynamic therapy of tumours. J Photochem Photobiol B 39:1-18.

Olivier D, Douillard S, Lhommeau I, Bigot E, Patrice T. 2009a. Secondary oxidants in human serum exposed to singlet oxygen: the influence of hemolysis. Photochem Photobiol Sci 8:1476-1486.

Olivier D, Douillard S, Lhommeau I, Patrice T. 2009b. Photodynamic treatment of culture medium containing serum induces long-lasting toxicity in vitro. Radiat Res 172:451-462.

Nariharan PV, Courtney J, Eleczko S. 1980. Production of hydroxyl radicals in cell systems exposed to haematoporphyrin and red light. Int J Radiat Biol 37:691-694.

Redmond RW, Gamlin JN. 1999. A compilation of singlet oxygen yields from biologically relevant molecules. Photochem Photobiol 70:391-475.

Reischl E, Dafre AL, Franco JL, Wilhelm Filho D. 2007. Distribution, adaptation and physiological meaning of thiols from vertebrate hemoglobins. Comp Biochem Physiol C Toxicol Pharmacol 146:22-53.

Schleicher E, Friess U. 2007. Oxidative stress, AGE, and atherosclerosis. Kidney Int Suppl S17-S26.

Seyama A. 1993. The role of oxygen-derived free radicals and the effect of free radical scavengers on skeletal muscle ischemia/reperfusion injury. Surg Today 23:1060-1067.

Sheldon J, Hoover JP, Payton ME. 2007. Plasma uric acid, creatinine, and urea nitrogen concentrations after whole blood administration via the gastrointestinal tract in domestic pigeons (Columba livia). J Avian Med Surg 21:130-134.

Snyder JW, Skovsen E, Lambert JD, Poulsen L, Ogilby PR. 2006. Optical detection of singlet oxygen from single cells. Phys Chem Chem Phys 8:4280-4293.

Sohal RS, Sohal BH, Brunk UT. 1990. Relationship between antioxidant defenses and longevity in different mammalian species. Mech Ageing Dev 53:217-227.

Summa D, Spiga O, Bernini A, Venditti V, Priora R, Frosali S, Margaritis A, Di Giuseppe D, Niccolai N, Di Simplicio P. 2007. Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions:The albumin model. Proteins 69:369-378.

Tanielian C, Mechin R, Seghrouchni R, Schweitzer C. 2000. Mechanistic and kinetic aspects of photosensitization in the presence of oxygen. Photochem Photobiol 71:12-19.

Thorington RW, Anderson S (1984). Primates. In: Anderson S, ed. Orders and Families of Recent Mammals of the World. New York: John Wiley & Sons. p 187-217.

Weishaupt KR, Gomer CJ, Dougherty TJ. 1976. Identification of singlet oxygen as the cytotoxic agent in photoinactivation of a murine tumor. Cancer Res 36:2326-2329.

Wicher KB, Fries E. 2006. Haptoglobin, a hemoglobin-binding plasma protein, is present in bony fish and mammals but not in frog and chicken. Proc Natl Acad Sci U S A 103:4168-4173.

Wright A, Hawkins CL, Davies MJ. 2003. Photo-oxidation of cells generates long-lived intracellular protein peroxides. Free Radic Biol Med 34:637-647.