Survival Study of Metallothionein-1 Transgenic Mice and Respective Controls (C57BL/6J): Influence of a Zinc-Enriched Environment

Introduction

M etallothioneins (MTs) are ubiquitous proteins that participate in the cellular response to numerous forms of stress. These proteins were originally thought to be “simple” regulators of metal homeostasis, but recent literature suggests that they are involved in key mechanisms associated with longevity. MTs can serve principally as a scavenger of free radicals, ¹ which are viewed as a driving force behind multiple age-associated pathologies. Modulation of mitochondrial respiratory function, ² inflammatory, ³ immune response, ⁴ brain repair and regeneration, ⁵ and cellular senescence ⁶ are among the functions currently attributed to MTs, with strong implication in aging processes. Their involvement in longevity emerges also from studies in long-lived worms, ⁷ cardiac-specific MT-2–overexpressing mice, ⁸ as well as from two association studies in humans. ^9,10 However, MTs have still not received appropriate attention in the context of aging biology and the study of basic aging mechanisms. Reviews covering potential downstream mechanisms of caloric restriction (CR) and/or insulin/insulin-like growth factor-1 (IGF-1) (IIS) signaling, for example, have not commonly discussed MTs as a target of potential importance.

Evidence has now shown that MT is induced by CR and IIS inhibition in invertebrate systems and mice. ¹¹ A recent meta-analysis of microarray data revealed that expression of MT-1 is elevated in hepatic tissue obtained from multiple long-lived dwarf mouse models. ¹² Taken together, these properties make MT an appealing research focus from the biogerontologist's perspective. However, observational studies in humans have also led to the hypothesis that MTs might display a potential antagonistic pleiotropic role that could be affected by nutritional zinc. ⁴ To gain further insight regarding the role of MTs in aging and longevity, we decided to study the survival of old MT-1–overexpressing mice (MT-TG) characterized by Iszard ¹³ and their respective controls (C57BL/6J) in normal breeding conditions and in presence of zinc (Zn)-supplemented water. The basic features shown by MT-TG mice seem to provide potential for a “robust” mouse model with delayed onset of age-related pathology, but up to now no focused research on longevity of these mice has been performed.

Material and Methods

Results

Median life spans for subgroups A, Az, B, and Bz were 710, 724, 839, and 824 days, respectively. Maximum life spans (measured as the age at death of the last survivor) for subgroups A, Az, B, and Bz were 895, 997, 995, and 1,016 days, respectively. Kaplan–Meier survival analysis, using pairwise comparison by log rank, Breslow, and Tarone–Ware tests did not show any significant difference between control and Zn-supplemented mice, both for the C57BL/6J and MT-TG strains. Conversely, significant differences (p<0.001) were observed when survival curves of C57BL/6J (supplemented or not) were compared with those from the MT-TG strains (supplemented or not) independently by the test used for pairwise comparison. Estimation of ω and S0 parameters using the Piantanelli model yielded the following results (expressed as mean±standard deviation [SD]): Group A, ω=0.032216±0.000093, S0=0.374752±0.005956; Group Az, ω=0.030875±0.000102, S0=0.428312±0.003599; Group B, ω=0.027950±0.000035, S0=0.309806±0.004039; Broup Bz, ω=0.028538±0.000034, S0=0.317393±0.004577. These results show that Zn treatment induced a 14% increase of S0 in the C57BL/6J mice, which is significant by observing the overlap between the 95% confidence interval of the estimated parameters (p<0.01). The 13% difference in the ω parameter between control C57BL/6J and MT-TG mice was also significant (p<0.01). Survival functions fitted with the Piantanelli model are shown in Fig. 1.

OPEN IN VIEWER

FIG. 1.

Survival functions fitted by the Piantanelli model of MT-TG and C57BL/6J mice treated with or without zinc (Zn). (Solid blue line with filled circles) C57BL/6J without Zn (ω=0.032216±0.000093, S0=0.374752±0.005956); (dashed blue line with open squares) C57BL/6J supplemented with Zn (ω=0.030875±0.000102, S0=0.428312±0.003599); (solid red line with filled circles) MT-TG without zinc (ω=0.027950±0.000035, S0=0.309806±0.004039); (dashed red line with open squares) MT-TG supplemented with zinc (ω=0.028538±0.000034, S0=0.317393±0.004577). Estimated parameters ω and S0 are reported as mean±standard deviation (SD). (Color image is available online at www.liebertonline.com/rej).

Discussion

This preliminary study was aimed mainly at observing the influence of a Zn-enriched environment on the median and maximum life span of normal C57BL/6J and “metal resistant” MT-TG mice. It was expected that the large dose of Zn administered for the whole life span starting from old age could have had a negative impact on life span, especially in the normal C57BL/6J strain. Indeed, it is known that large doses of Zn could display toxic effects, and that mice overexpressing MTs are more resistant to treatment with toxic doses of metals. ¹⁷ However, survival analysis by Kaplan–Meier failed to show any significant difference between supplemented and nonsupplemented groups. Conversely, the analysis of survival data with the Piantanelli parametric model suggests that the treatment is able to affect the survival curve of the C57BL/6J mice. The model shows a large difference in the S0 parameter between supplemented and not supplemented C57BL6 mice (≈14%) that deserve some considerations.

The advantage of using a parametric model is the possibility of obtaining a biological meaning from a simple survival analysis. S0 describes the extent of the variability of physiological functions due to combined genetic differences and environmental fluctuations in space and time. The observed differences in S0 suggest that the treatment does not influence all mice in a completely identical way. In other words, the increased S0 suggests that some mice benefit whereas others are negatively influenced by the changed conditions, thus leading to an increased heterogeneity within the population. These changes in S0 were no more evident between supplemented and “not supplemented” MT-TG-mice (≈2.4% difference).

Therefore, it might be hypothesized that Zn treatment might influence the survival of an individual mouse, depending on its ability to produce MT in response to Zn supplementation. Although the mice should be genetically identical, it has been demonstrated that they could display important variation in gene expression that could be attributed to stochastic and microenvironmental factors. ¹⁸ These differences could explain the phenotypic consequences of Zn supplementation observed in this study. To confirm this hypothesis, it would be needed to repeat the survival analysis while measuring MTs using noninvasive methods.

The data presented in this form open an interesting perspective in the biological assessment of parameters that are associated with a better or worse response to zinc supplementation in animal models with a potential for translation in humans. This response could be related to the heterogeneous induction of MT in response to Zn treatment that could be different, even among the same strain. ¹⁹ Moreover, although the difference in ω was relatively scarce between supplemented and nonsupplemented groups, we observed a large difference (≈13%) between the C57BL/6J and the MT-TG mice strains. These last data are in agreement with the view that improving MT response may be an interesting approach to extended longevity and health span. Om the other hand, the large difference in the median life span observed between the MT-TG strain (839 days for nonsupplemented and 824 for the supplemented mice) and the C57BL/6J strain (710 for the nonsupplemented and 724 for the supplemented mice) is just enough to suggest the relevance of these proteins in counteracting age-associated morbidity and mortality.

It is important to consider that, although all mice were kept in identical conditions, the animals were not kept in a pathogen-free environment. These conditions, although more similar to the human environment, could have affected the survival curves of the laboratory strains, as the median and maximum life span of our C57BL/6J mice were slightly shorter than those reported by others. ²⁰ Therefore, the results presented here on the increased maximum life span of our MT-TG mice compared to normal C57BL/6J should be considered with careful attention. The large increase in median life span of our MT-TG strains compared to the C57BL/6J strain suggests that improving MT production might have an important role to reduce morbidity in old animals. These data confirm the relevance of MT in aging and suggest that further trials using MT gene therapy should be considered for its effect on mortality and morbidity in old animals.