Bomb pulse biology

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Abstract

The past decade has seen an explosion in use of the 14C bomb pulse to do fundamental cell biology. Studies in the 1960s used decay counting to measure tissue turnover when the atmospheric 14C/C concentration was changing rapidly. Today bulk tissue measurements are of marginal interest since most of the carbon in the tissue resides in proteins, lipids and carbohydrates that turn over rapidly. Specific cell types with specialized functions are the focus of cell turnover investigations. Tissue samples need to be fresh or frozen. Fixed or preserved samples contain petroleum-derived carbon that has not been successfully removed. Cell or nuclear surface markers are used to sort specific cell types, typically by fluorescence-activated cell sorting (FACS). Specific biomolecules need to be isolated with high purity and accelerator mass spectrometry (AMS) measurements must accommodate samples that generally contain less than 40 μg of carbon. Furthermore, all separations must not add carbon to the sample. Independent means such as UV absorbance must be used to confirm molecule purity. Approaches for separating specific proteins and DNA and combating contamination of undesired molecules are described.

Introduction

The near doubling of the atmospheric 14CO2 concentration from 1955 to 1963 due to atmospheric above ground testing of nuclear weapons and its subsequent decline is documented by a high resolution record [1], [2], [3], [4], [5]. This rapid rise, sharp peak, and exponential decline is known as the 14C bomb pulse. Most of the tests occurred at relatively few sites in the Northern Hemisphere and it took several years for the excess 14C to distribute homogeneously throughout the atmosphere [2]. The excess 14CO2 entered the food chain through photosynthesis and completely labeled the terrestrial biosphere. The variation in the 14C provides an isotopic chronometer of molecular biosynthesis since 1955. This paper describes the use of the bomb pulse as a tracer in cell and molecular biology.

The elevation of 14C in the biosphere and humans that occurred due to above ground nuclear weapons testing was documented almost immediately [6], [7]. Throughout the 1960s and 1970s the rise and fall of 14C/C in the biosphere continued to draw attention [8], [9], [10], [11]. Since the 14C/C level was changing rapidly in the 1960s, it was relatively straightforward to determine carbon turnover rates of many tissues [8], [9], [10], [11]. These studies used 14C decay counting and required grams of carbon from tissue samples for analyses. Different cell types were also not separated in these tissues due to sample size requirements.

The development of AMS in the late 1970s enabled analyses of much smaller samples and examination of carbon turnover in specific biological structures. Initially researchers concentrated on forensic applications of bomb pulse. The first biological application by Druffel and Mok dated gallstones [12], containing significant amounts of organic material (mostly cholesterol and bile pigments) from patients who lived in the northern hemisphere. The next biological application of the bomb pulse involved separating human lung parenchymal elastic fibers that were determined to be as old as the individual using aspartate racemization and 14C analyses [13]. The longevity of the elastin was suspected at the time, but nobody had previously thought of using the bomb pulse to probe human tissue turnover in this manner. Bomb pulse dating was largely ignored in the biological community for the next several years, until the pathological structures associated with Alzheimer’s disease, neurofibrillary tangles (NFT) and senile plaques (SP), were separated gravimetrically and analyzed for 14C/C post mortem using healthy tissue as controls [14]. The ages of the structures were averages accumulated over the lifetimes of the donors. In half the cases examined, the average ages of the SP and NFT predated the onset of clinical symptoms of Alzheimer’s disease while the other half possessed average SP and NFT ages corresponding to the onset of symptoms or younger [14]. The lens of the eye also possesses long-lived proteins. Lynnerup et al. showed that the proteins of the lens had very limited turnover or repair [15]. Recent studies of arterial plaques show that the deposits that clog arteries accumulate over many years [16], [17].

A leap occurred in biological analysis with the 14C bomb pulse when the analysis of 14C/C in DNA to ascertain cell birth date was first performed. Since DNA only acquires significant new carbon at cell division, the 14C/C of genomic DNA is a time stamp of cell birth date [18]. Genomic DNA dating has been used to confirm that neurogenesis does not occur in the human cerebellum [18] or cortex [19] after birth. The lack of bomb pulse carbon in neuronal DNA of subjects born before 1955 indicated that DNA repair provides an insignificant amount of new carbon after cell division [18]. It has also been used to determine that adipocytes turnover approximately every 10 years [20] and cardiomyocytes turnover at a low rate [21]. Using bromodeoxyuridine (BrdU) and 14C/C analyses of DNA from pancreatic β-cells, it was determined insulin producing β-cells turnover at a 1–2% annual rate through early adulthood and then cease to turnover after age 30 [22].

Section snippets

Methods

Researchers must obtain approval from local Human Subject Ethics Boards before embarking on a bomb pulse study. Each country has its own specific laws governing human subject research based on the Helsinki Declaration [23] that is an update and expansion of principles from the Nuremberg Code [24]. Human subjects in bomb pulse biology studies are often deceased as tissues are most often obtained from autopsies or tissue banks. Permission to harvest tissues for research must be obtained from

Constructing models

When turnover occurs, a model must be constructed to account for the difference between the 14C/C at the time of birth and its value at measurement [45]. More than one model can be constructed from the same set of data, and multiple scenarios should be evaluated. When determining the age of a deposit or pathological structure, elevation of 14C/C compared to the contemporary level at harvest gives an average age of the structure. By using subjects of different ages, a model of cell turnover can

Conclusion

Bomb pulse dating in cell and molecular biology is challenging. Obtaining clean separations of specific biomolcules has been the most difficult task. Every system is different, so sorting parameters change among cell types. We have found that markers touted as specific in the literature can be generic, common to many cell types. Samples tend to be small and more challenging to analyze by AMS than typical samples. Small samples are more sensitive to contamination so relevant backgrounds and

Acknowledgements

Support was provided by Grants from the National Center for Research Resources (5P41RR013461-14) and the National Institute of General Medical Sciences (8P41GM103483-14) from the National Institutes of Health, NEI 5R21EY018722 and LLNL LDRD (10-LW-033). This work performed under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

References (49)

  • M.A. Lovell et al.

    Neurbiol. Aging

    (2002)
  • K.L. Spalding et al.

    Cell

    (2005)
  • A. Pearson et al.

    Geochim. Cosmochim. Acta

    (2001)
  • R.H. Smittenberg et al.

    J. Chromatogr. A

    (2002)
  • K. Alkass et al.

    Forensic Sci. Inter.

    (2011)
  • M. Stuiver et al.

    Radiocarbon

    (1998)
  • Q. Hua et al.

    Radiocarbon

    (2004)
  • I. Levin et al.

    Radiocarbon

    (2004)
  • H.D. Graven et al.

    J. Geophys. Res.-Atmos.

    (2012)
  • I. Levin et al.

    Tellus

    (2010)
  • T.A. Rafter et al.

    Science

    (1957)
  • W.S. Broecker et al.

    Science

    (1959)
  • W.F. Libby et al.

    Science

    (1964)
  • R. Nydal et al.

    Nature

    (1971)
  • D.D. Harkness et al.

    Nature

    (1972)
  • M.J. Stenhouse et al.

    Nature

    (1977)
  • E.M. Druffel et al.

    Radiocarbon

    (1983)
  • S.D. Shapiro et al.

    J. Clin. Invest.

    (1991)
  • N. Lynnerup et al.

    PLoS ONE

    (2008)
  • I. Gonçalves et al.

    Circ. Res.

    (2010)
  • S. Hägg et al.

    PLoS ONE

    (2011)
  • R.D. Bhardwaj et al.

    PNAS

    (2006)
  • K.L. Spalding et al.

    Nature

    (2008)
  • O. Bergmann et al.

    Science

    (2009)
  • Cited by (0)

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