Effects of Thermal Stress on Hepatic Melanomacrophages of Eupemphix nattereri (Anura)
Lia Raquel De Souza Santos,
Goiano Federal Institute (IFGoiano) campus RioVerde, Goiás, Brazil
Correspondence to: Lia Raquel de Souza Santos, Instituto Federal Goiano—IFG/Campus Rio Verde—GO, Rodovia Sul Goiana Km 01—Zona Rural, Cep: 75.901–970—Rio Verde, GO, Brasil. E-mail: email@example.com
The hematopoietic organs of ectothermic animals commonly display pigmented cells with phagocytic activity called melanomacrophages or Kuppfer cells (Sichel et al., 1997, Corsaro et al., 2000; Prelovsek and Bulog, 2003). These cells are resident macrophages of the liver that originate from the hematopoietic stem cells (Gallone et al., 2002; Sichel et al., 2002). They can produce and store melanin, which absorbs and neutralizes free radicals, cations, and other potentially toxic agents, derived from the degradation of phagocytized cellular material (Zuasti et al., 1989; Agius and Roberts, 2003; Franco-Belussi et al., 2013).
In addition to melanin, two other compounds are found in melanomacrophages, both derived from cell catabolism: hemosiderin, originating from the degradation of erythrocytes (Kranz, 1989) and lipofuscin, resulting from the oxidative polymerization of polyunsaturated fatty acids. In fish, lipofuscin seems to be related to deficient diets (Pickford, 1953). These substances are variable depending on the physiological status of anurans (Franco-Belussi et al., 2013).
The liver pigment cells show high metabolic and cytokinetics abilities during the annual cycle of the frog (Barni et al., 2002). In the winter, temperature, photoperiod and nutritional status, can promote changes in cellular mechanism, resulting in increased pigmentation in this organ (Corsaro et al., 2000; Barni et al., 2002). The hepatic pigmentation is also related with respiratory conditions in newts, where the genetic expression of tyrosinase is increased under hypoxia. This physiological response prolongs survival time under anaerobic conditions because it reduces the metabolic rate and melanin seems to be a byproduct of this mechanism (Frangioni et al., 2000).
Amphibians have a highly permeable skin and a water-dependent life cycle. For this reason, they are very susceptible to aquatic pollutants, global warming and other environmental changes (Blaustein and Wake, 1990; Alford and Richards, 1999; Blaustein et al., 2011, 2003). Since temperature can cause liver metabolic alterations (Corsaro et al., 2000; Barni et al., 2002), including changes in melanin-containing cells, this study aims at performing morphologic and histochemical analysis of the Eupemphix nattereri hepatic pigmentation after hyperthermic and hypothermic stress. Therefore, we hypothesize that pigmentation will change due to thermal stress.
MATERIAL AND METHODS
We collected 65 Eupemphix nattereri adult males during the reproductive period (from November 2007 to February 2008) from temporary and permanent ponds in São José do Rio Preto-SP, (20°49′12.65″S and 49°22′46.85″W) Southeastern Brazil, with permission granted by IBAMA (RAN/IBAMA 18573-1). The animals remained in the terrarium (73 × 41 × 52 cm) during 7 days where they received water and food. Animal handling and experiments were performed according to the ethical guidelines of the São Paulo State University (Protocol 70/07-CEEA).
Thermal Variation Experiments
Thermal stress was induced using a B.O.D. incubator (Model 121 FC) where the animals were divided into two groups of 30 animals each and submitted to hypothermic and hyperthermic conditions. Hyperthermia (35.1°C) and hypothermia (18.9°C) were induced by rising or reducing, respectively, the room temperature (27°C) by 30%. To determine the temperature, we considered two factors: the average temperature recorded three times a day during animal stay in the laboratory and the annual average temperature recorded during the year before the experiments. In this way, we tried to determine the minimum and maximum temperature variation tolerated by the animals.
Thirty frogs were separated into three groups of 10 animals, which remained in the BOD incubator at 35.1°C for a period of 12, 24, and 48 hr, respectively, to check the effects of elevated temperature on pigmented cells. At the end of each period, five samples from each group were analyzed immediately while the other five animals were removed from the BOD incubator and kept in the recovery room at 27°C temperature for the same period of time they spent in the incubator and were subsequently analyzed.
Thirty frogs were separated into three groups of 10 animals, which remained in the BOD incubator at 18.9°C for a period of 12, 24, and 48 hr, respectively, to check the effects of low temperature on pigmented cells. At the end of each period, five samples from each group were analyzed immediately while the other five animals were removed from the BOD incubator and kept in the recovery room at 27°C temperature for the same period of time they spent in the incubator and were subsequently analyzed.
For the control group five animals were maintained under the same conditions, when at room temperature.
Morphological and Histochemical Analyses
The liver fragments were fixed in Karnovsky solution (Sörensën phosphate buffer 0.1 M, phosphate buffer pH 7.2 with 5% paraformaldehyde and 2.5% glutaraldehyde) for 24 hr at 4°C. Then, the samples were washed in distilled water, dehydrated in alcohol series and embedded in historesin (Leica-historesin embedding kit). Histological 2-µm sections were obtained using a microtome (Leica RM 2265), stained with Hematoxylin-eosin and analyzed using a Leica DM4000 B microscope.
Histological sections of liver were incubated for 15 min in Schmorl's solution for lipofuscin histochemistry. For detection of hemosiderin, samples were incubated in acidic ferrocyanide, followed by neutral red and eosin solutions.
We analyzed ten animals for each experimental group (12 hr, 24 hr, and 48 hr) as follows: five immediately after thermal stress (I) and five after a recovery period (R). One hundred fifty histological parts were analyzed for each experimental group at different times, for the purpose of determining the relation between pigmentation and hepatic temperature variation. The Image Pro-Plus (Media-Cybernetics Inc., version 4.5) software was used to quantify the pigmentation area. The quantification technique is based on different color observed in the histological liver parenchyma, stained with hematoxylin-eosin, according to a modified method initially described Lehr et al. (1997).
We tested if the pigmented area occupied by melanin, hemosiderin and lipofuscin (response variable) increased with thermic stress exposure time (predictor variable). One-way ANOVA was used, followed by a post hoc Tukey test for parametric data. The nonparametric data were square root transformed to meet the assumptions of variance normality and homogeneity. When the data remained nonparametric, the Kruskal-Wallis test was used followed by a post hoc test. Analyses were conducted using the R version 2.11.1 software (R Development Core Team, 2010).
Melanomacrophages were observed among the double cordon of hepatocytes, near the sinusoids (Fig. 1A,B). We also observed hemosiderin and lipofuscin within melanomacrophages (Fig. 1C,D). The results showed that liver pigmentation decreases with thermal stress; the hypothermic conditions led to a rapid decrease, but no differences were found between the treatments (immediately or recovered). However, under hyperthermic conditions, pigmentation decreased 24 hr after stress, with differences between treatments.
The hepatic pigmentation decreased under hyperthermic conditions in specimens analyzed immediately after thermal stress (F = 10.74; P ≤ 0.01). Although statistical differences were found between the 12 and 24-hr groups (F = 10.74; P ≤ 0.05), neither group was different from the control group (F = 10.74; P ≥ 0.05). Pigmentation increased gradually in the recovered specimens (24 and 48 hr), in which the pigmented area was similar to that in the control group (F = 31.61; P ≥ 0.05) (Fig. 2).
When we compared the effects of the treatments (immediately and recovered) in each experimental time separately, we observed a decrease in hepatic pigmentation in specimens recovered after 12 hr, with a significant difference between the treatments (F = 34.35; P ≤ 0.01). The pigmented area did not differ in specimens analyzed immediately after 24 hr of hyperthermic conditions when compared with the recovered ones (F = 3.46; P ≥ 0.05). The hepatic pigmentation decreased in specimens analyzed immediately after 48 hr, when compared with the control and the recovered specimens (F = 15.41; P ≤ 0.01) (Fig. 2).
Hemosiderin decreased under hyperthemic conditions in specimens analyzed immediately after 12 and 48 hr of thermal stress (F = 24.47; P ≤ 0.01; P = 0.002), and after 12 hr of recovery (F = 32.43; P ≤ 0.01) (Fig. 2B). This substance also decreased, as shown by the comparison of the treatments (immediately and recovered) after 12 and 24 hr (F = 21.99; P ≤ 0.01; F = 13.49; P ≤ 0.01); however, after 48 hr, the hemosiderin levels in the recovered group had increased compared with the immediate group (F = 33.75; P ≤ 0.01) (Fig. 3).
The lipofuscin area increased under hyperthermia conditions immediately after 12 and 24 hr of stress (F = 12.34; P ≤ 0.01; F = 12.34; P = 0.003). However, the animals recovered from the stress, showed decreased lipofuscin levels after 12 hr (F = 20.63; P = 0.006) but increased levels after 48 hr (F = 12.34; P ≤ 0.01) (Fig. 2C). compared with the experimental treatments (immediately and recovered) at the same timepoint, we observed a decrease in the animals recovered from 12 and 24 hr (F = 39.61; P ≤ 0.01; F = 38.76; P ≤ 0.01, respectively) and an increase in the animals recovered from 48 hr (F = 18.30; P ≤ 0.01) (Fig. 4).
Hepatic pigmentation decreased in specimens analyzed immediately after exposure to hypothermic conditions compared with the control (F = 6.04; P ≤ 0.05). However, the amount of pigmentation present in specimens analyzed at different times was similar (F = 6.04; P ≥ 0.05). The hepatic pigmentation of recovered specimens decreased after 12 hr and 24 hr compared with the control group (H = 13.89; P ≤ 0.05). Hepatic pigmentation increased in the 48 hr-recovered group and was similar to the control (H=13.89; P ≥ 0.05) (Fig. 5).
Comparison of the effects between treatments shows that the amount of pigmentation was similar for all experimental periods (12 hr, 24 hr, and 48 hr) (F = 6.70; P ≥ 0.05). However, the pigmentation in both treatments was lower for the 12 hr and 24 hr groups compared with the control (12 hr: F = 6.70; P ≤ 0.05; 24 hr: F = 9.21; P ≤ 0.01). Hepatic pigmentation was similar in the specimens analyzed immediately after thermal stress and in the recovered ones in the 48 hr period (H=9.17; P ≥ 0.05). However, liver pigmentation tended to increase in the specimens analyzed immediately after hypothermia (48-hr group), remaining similar to control (H=9.17; P ≥ 0.05) (Fig. 5).
Under hypothermic conditions, hemosiderin did not vary for animals analyzed immediately (F = 2.08; P = 0.10) after thermal treatment, nor for recovered animals (F = 2.62; P = 0.05) (Fig. 6). Similarly, lipofuscin did not vary under hypothermic conditions for both animals analyzed immediately after 12, 24 and 48 hr (F = 0.87; P = 0.46) and recovered animals in the 12 and 48 hr groups after thermic stress (F = 3.00; P = 0.38), but the lipofuscin area increased in animals recovered after 24 hr (F = 3.00; P = 0.03) (Fig. 7).
Interactions Between Hyperthermia and Hypothermia Treatments
The immediate effects of thermal stress in the experimental groups were different only in the specimens analyzed after 12 hr, with a decrease in the pigmented area under hypothermic conditions (F = 6.67; P ≤ 0.05). The hyperthermic and hypothermic conditions did not result in significant pigmentation differences at other experimental times (24 hr: F = 6.63; P ≥ 0.05; 48 hr: F = 10.91; P ≥ 0.05). However, all experimental groups (12 hr, 24 hr, and 48 hr) showed a decrease in hepatic pigmentation when compared with control (F = 6.04; P ≤ 0.05), under hypothermia. As temperature increased, no significant differences were observed between animals analyzed immediately after 12 hr compared with the control. Under hyperthermia, the animals analyzed after 24 hr showed a tendency to decrease the pigmented area, although similar to both control and experimental period of 48 hr (F = 10.74; P ≥ 0.05) (Fig. 8A).
Under hypothermic conditions, the hemosiderin area in animals immediately after the treatments decreased compared with hyperthermic groups after 12 and 48 hr of stress period (F = 9.65; P = 0.01; P = 0.04, respectively) (Fig. 8B). However, the lipofuscin area increased under hyperthermia compared with hypothermia in the animals of the 12 and 24-hr groups (F = 7.35; P < 0.01; P = 0.04, respectively) (Fig. 8C).
After thermal stress, the recovered specimens showed statistical differences between all experimental times (12 hr: F = 19.03; P ≤ 0.05; 24 hr: F = 8.94; P ≤ 0.01; 48 hr: H=6.69; P ≤ 0.05). Hepatic pigmentation decreased in the two 12 hr experimental groups (hyperthermia: F = 31.61; P ≤ 0.01; hypothermia: F = 6.04; P ≤ 0.05). Pigmentation decreased after 24 hr under hypothermia (24 hr: H=13.89; P ≤ 0.05). The pigmentation in the 48 hr hyperthermia group was similar to that in the control (Fig. 8A). Hemosiderin and lipofuscin decreased in the 12 hr hyperthermia recovery group (F = 14.63; P < 0.01; F = 10.09; P < 0.01, respectively) (Fig. 8B,C) and remained unchanged after 24 hr (F = 14.63; P = 0.94; F = 10.09; P = 0.99, respectively) (Fig. 8B,C). After 48 hr, the lipofuscin area under hyperthermia increased compared with hypothermic conditions (F = 10.09; P = 0.03) (Fig. 8C).
Thermal stress was found to decrease hepatic pigmentation, suggesting metabolic changes in the melanomacrophages of the liver of Eupemphix nattereri. These cells are very plastic and can respond to seasonal variations (Barni et al., 1999). Thus, it appears that these cells are sensitive to stimuli as a way to adapt to stress conditions. Frangioni et al. (2000) showed that the variation in the amount of hepatic melanin is related to temperature, since melanin was less abundant in low temperatures (about 6°C; hibernation temperature) than in warm ones (about 22°C). In addition, Guida et al. (1998) found evidence of melanogenic activity in cultured melanomacrophages and found that these cells had an internal molecular clock in the first 72 hr of cell culture. However, our data are in accordance with those of Barni et al. (2002), who showed that the decrease in hepatic pigmentation is related to seasonal variations. This decreased pigmentation is due to a decreased number of melanosomes by autophagy and the synthesis of melanin is due to apoptotic mechanisms.
The decreased liver pigmentation under hyperthermic conditions can be attributed to the function of melanin in the thermoregulation system, since it absorbs radiation and transforms it into heat (Césarini, 1996). As melanin is one of the components of melanomacrophages, the decrease in pigmentation causes a reduction in energy absorption and, consequently a reduction in heat absorption by hepatocytes. Thus, the decrease in pigmentation probably occurs to maintain homeostasis by means of thermoregulation. This decrease results from the inactivation of the protein phosphates, which promotes several changes that inhibit the transcriptional regulator and the synthesis of melanin (Kim et al., 2005). Furthermore, tyrosine activity decreased in hepatic melanomacrophages of R. esculenta in the summer, when temperatures are higher (Cicero et al., 1989). Therefore, we hypothesize that the decrease in liver pigmentation in E. nattereri in hyperthermic stress conditions is related to decreased tirosinase activity.
In hypothermia, we also observed a decrease in hepatic pigmentation, since it is related to the inhibition of tyrosinase, which in turn reduces the synthesis of melanin (Kim et al., 2003). At 27°C, the synthesis of melanin was not affected (Kim et al., 2003), therefore the return to room temperature was responsible for an increase in hepatic pigmentation of specimens in the two experiments. However, Corsaro et al. (1990) reported that melanin in hepatic melanomacrophages in R. esculenta increased during the winter (about 5–10°C) and decreased during the summer (about 20–25°C). The decreased pigmentation we found can be explained by the temperature used (18.9°C; winter temperature of the study region), which is higher than the winter temperature of a temperate climate.
According to Tonosaki et al. (2004), in ectothermic animals, drastic changes in ambient temperature lead to physiological changes, brought about by releasing or inhibiting the Melanin Stimulating Hormone (α-MSH) as a response to stress. In constrast, a gradual physiological response is observed when the variation in the environmental temperature is also gradual. Thus both experiments showed that hepatic pigmentation is influenced not only by thermal stress, but also by the time of exposure.
Hemosiderin and lipofuscin are substances that originate from cellular catabolism and vary in number in conditions of thermal stress. Hemosiderin is known to decrease with hyperthermia, which is related to a decrease in the catabolism of damaged erythrocytes (Kranz, 1989), whereas lipofuscin, which is produced by destabilization of cellular membranes, possibly as a result of thermal stress, increases during hyperthermia (Terman and Brunk, 2004; Kurz, 2008).
When comparing the effects of hyperthermia and hypothermia, we found that increased temperatures rather than hypothermic conditions led to drastic physiological changes. We hypothesize that E. nattereri is less tolerant to temperature variations, since it occurs a tropical climate, in which the temperature variation is lower than in a temperate climate. As a result, a few degrees of variation in temperature are sufficient to induce changes in liver pigmentation of E. nattereri. Therefore, these results may have broader implications, such as detect direct or indirect environmental effects, particularly climate change, degradation of natural habitat, and long-term consequences of global warming. Conversely, some species are more sensitive to this variation. In this study, morphological aspects can be related to environmental degradation.
In conclusion, this study showed that hepatic pigmentation in anurans is affected by temperature variation. The increase in temperature rather than hypothermia causes drastic physiological changes. The body also has more difficulty restoring its normal status after hyperthermia. Our results demonstrate that thermal stress, is able to compromise the morphology and liver function. Thus, increased/decreased liver pigmentation is related to environmental factors, such as temperature variation, to which these organisms are susceptible. Our findings can be used as biomarkers for morphological environmental effects.
The authors are grateful to Dr. Vitor Hugo M. Prado and to Diogo Borges Provete for their helpful suggestions on the article. R.M.V.K. for revising manuscript language.