A variety of living organisms has been found recently that are biochemically able to precipitate the ferromagnetic mineral magnetite (Fe3O4). Originally discovered in the radular teeth of a primitive marine mollusc (Lowenstam, 1962), magnetite has since been reported in bacteria (Frankel, Blakemore & Wolfe, 1979), arthropods (Gould, Kirschvink & Deffeyes, 1978), and vertebrates (Walcott, Gould & Kirschvink, 1979; Zoeger, Dunn & Fuller, 1981; Walker & Dizon, 1981). Although the presence and biological origin of this material are clear, very little is yet known about the distribution or metabolic function of ferromagnetic minerals in vertebrate tissue. Magnetic remanence, which uniquely indicates the presence of ferromagnetic particles, has been previously detected in localized areas associated with the dura membranes of homing pigeons (Walcott et al. 1979) and dolphins (Zoeger et al. 1981), in pigeon neck muscles (Presti & Pettigrew, 1980), in the mid-brain of monkeys, and in human adrenal glands (Kirschvink, 1981). We report here the first discovery of anomalously high concentrations of ferromagnetic material in two strains of neoplasms, YC-8 lymphoma and Lewis lung tumour, as well as the apparent absence of such material in three human carcinomas (gastric, colon and renal)

Batkin, Stanley

Kirschvink, Joseph L.

Tabrah, Frank L.

English

Caltech Authors - Main

J. exp. Biol. (1982), 101, 331-3*6 321
ittth i figure
Printed in Great Britain
SHORT COMMUNICATION
FERROMAGNETISM IN TWO MOUSE TUMOURS
BY JOSEPH L. KIRSCHVINK
Division of Geological and Planetary Sciences,
The California Institute of Technology, Pasadena, California
FRANK L. TABRAH
Cancer Center of Hawaii, School of Medicine,
University of Hawaii, Honolulu, Hawaii
AND STANLEY BATKIN
Cancer Center of Hawaii, School of Medicine,
University of Hawaii, Honolulu, Hawaii
(Received 1 March 1982 - Accepted 30 March 1982)
A variety of living organisms has been found recently that are biochemically able
to precipitate the ferromagnetic mineral magnetite (Fe3O4). Originally discovered in
the radular teeth of a primitive marine mollusc (Lowenstam, 1962), magnetite has
since been reported in bacteria (Frankel, Blakemore & Wolfe, 1979), arthropods
(Gould, Kirschvink & Deffeyes, 1978), and vertebrates (Walcott, Gould & Kirschvink,
1979; Zoeger, Dunn & Fuller, 1981; Walker & Dizon, 1981). Although the presence
and biological origin of this material are clear, very little is yet known about the distri-
bution or metabolic function of ferromagnetic minerals in vertebrate tissue. Magnetic
remanence, which uniquely indicates the presence of ferromagnetic particles, has been
previously detected in localized areas associated with the dura membranes of homing
pigeons (Walcott et al. 1979) and dolphins (Zoeger et al. 1981), in pigeon neck muscles
(Presti & Pettigrew, 1980), in the mid-brain of monkeys, and in human adrenal glands
(Kirschvink, 1981). We report here the first discovery of anomalously high concen-
trations of ferromagnetic material in two strains of neoplasms, YC-8 lymphoma and
Lewis lung tumour, as well as the apparent absence of such material in three human
carcinomas (gastric, colon and renal).
Our previous studies revealed biological effects of non-ionizing radiation on tumour
cells (Batkin & Tabrah, 1977). In a trial study to attempt to enhance this effect, we
added paramagnetic spin-labelled compounds to the cells in vitro. Cell changes were
observed after the addition of the paramagnetic compounds alone (Batkin, Tabrah &
Misconi, 1980) and after further exposure to altered magnetic fields. Reports of
ferritin-bearing tumours (Gropp, Havemann & Lehmann, 1978; Hann et al. 1979;
Kew et al. 1978) and of magnetite in varied biological specimens led us to analyse
selected tumours in our laboratory for the presence of ferromagnetic material.
Cells of Lewis lung tumour and YC-8 lymphoma were maintained by repeated
•ibcutaneous transplant in female C57BL/6 and BALB/c mice. Experimental
•mours were excised from stock animals using non-magnetic glass microtome knives
322 J. L. KIRSCHVENK, F. L. TABRAH AND S. BATKIN
Table i. Ferromagnetic remanence in mouse tumours
Tumour tissue
YC-8 lymphoma
Sample A
Sample B
Lewis lung
Sample A
Sample B
sIRM
(PT)
840
1290
750
620
Sample
volume
(ml, ±10%)
0-44
0-18
1-71
0 8 6
Tumour cells
per sample
(±10%)
78x10'
3-1 xio1
10-9 x io'
5-5x1°'
Average remanence
per cell
(U/T]/cell)
4-7 x 10""
7-2 x io"17
H-8XI0-"
9-7 x 10""
The induced magnetization, termed a saturation Isothermal Remanent Magnetization (sIRM), is
the magnetic moment measured on the superconducting magnetometer in a zero field divided by the
sample volume. Although the saturation magnetization for magnetite is 48 milli-Tesla (mT), the satura-
tion remanence for a dispersion of single-domain crystals is half of this, or 24 mT. An electromagnetic
unit (e.m.u.) is 1 Gauss.cm1 or i o " ' J / T .
Note: The following human tissues were found to have no measurable remanence ( < io"1 0 J /T) ;
normal stomach (control), gastric carcinoma, colon carcinoma, and renal carcinoma. Control samples
of muscle and connective tissue from normal female C57BL/6 and BALB/c mice displayed only weak
saturation remanences of 32 and 45 pT respectively.
on the 13th day following transplant. Control samples of muscle and connective tissue
from both strains of mice were also taken for comparison. In addition, three human
carcinomas (gastric, colon and renal) were obtained after routine surgery in Honolulu.
All tissue samples for magnetic analysis were first thoroughly cleaned using ultrasonic
treatment in glass-distilled water. Using glass knives to avoid possible contamination
from metal instruments, each tumour was subdivided into two or more subfractions
of appropriate size for the SQUID magnetometer. All samples were frozen prior to
and during the magnetic analysis.
Magnetic measurements on these tissue samples were performed on a super-con-
ducting rock magnetometer of the type described by Goree & Fuller (1976), which
was housed in a magnetically shielded mu-metal room. To reduce the chance of
unwanted ferromagnetic contamination from dust particles during the measuring
procedure, a temporary clean-lab enviroment was made in the magnetometer area
using sheets of polyethylene plastic. A remanent magnetization was induced in each
sample prior to measurement by briefly exposing it to the field of a small CoSm
magnet (maximum intensity 0-3 Tesla, T). Although these magnets have an inhomo-
geneous field, most of the volume of each sample was exposed to intensities in excess
of o-i T, which is above the coercivity of all but the most highly elongate magnetite
crystals. Tissue samples which displayed a measurable ferromagnetic remanence
were then progressively demagnetized in an alternating magnetic field, using standard
paleomagnetic techniques (see McElhinny, 1973, for detailed discussions of these
methods). Ice-cube control samples made from glass-distilled water were magnetized
and measured along with the experimental tissues as a control for laboratory generated
contamination; no magnetic remanence was found in them. Estimates (± 10%) for the
number of tumour cells in the experimental tissues were then made from histological
sections, using a micrometer grid. In both the YC-8 lymphoma and Lewis lung tumour
samples, between 5 and 10% of the volume was composed of blood vessels and
connective tissue which was similar in appearance to that of the control samples.
sJRM-i
Ferromagnetism in two mouse tumours 323
Lewis lung Lymphoma
Peak alternating field (mT) Peak alternating field (mT)
Fig. 1. Progressive alternating-field demagnetization of tumour tissues. Each frozen sample
was initially exposed to the strong magnetic field of a CoSm magnet, producing a saturation
Isothermal Remanent Magnetization (sIRM), which was then measured on the super-
conducting magnetometer. Each sample was then sequentially demagnetized along three
perpendicular axea^starting with a 2-5 mT, 400 Hz field from a solenoid (Schonstedt Instrument
Co.) and remeasured. This procedure was repeated, using progressively stronger peak alterna-
ting fields until most of the remanence had been destroyed. An assemblage of identical,
non-interacting, single-domain grains treated in this fashion should produce a sigmoidal
curve similar to those observed here. This implies that within each rumour type the particles
are nearly identical, although YC-8 lymphoma and Lewis lung tumour form crystals of
different size and shape.
Results from the magnetic analysis are displayed in Table 1 and Fig. 1. All samples
of Lewis lung tumour and YC-8 lymphoma gave signals from 100 to 1000 times the
background noise on the superconducting magnetometer, whereas samples from the
normal mice and the human stomach, colon, and renal carcinomas were not notably
ferromagnetic. Per cell, Lewis lung tumour is on the average nearly twice as magnetic
as YC-8 lymphoma. Measurements on subdivided samples indicate that the magnetic
material has a nearly uniform distribution. The progressive demagnetization experi-
ments (Fig. 1) indicate that there are additional differences in the magnetic particles
between the two cell types. Most of the ferromagnetic crystals in Lewis lung tumour
have coercivities from 20 to 30 milli-Tesla (mT), whereas those in lymphoma cells
are below 15 mT. Normally, the magnetic moment of such a particle is parallel to its
long dimension, but it can point in either of two directions. For single-domain crystals,
the coercivity is essentially the external magnetic field strength necessary to flip the
direction of its remanent moment between these two positions. Although the coercivity
of a ferromagnetic crystal is a complex function of its composition, size and shape, it
generally increases with both particle elongation and volume. A comparison of these
results with fig. 50 of McElhinny (1973) suggests that the Lewis lung tumour crystals
are slightly larger or more elongate than those in YC-8 lymphoma cells, and that
cells within one strain have nearly identical magnetic particles. Magnetite crystals
g^ide by a pure strain of magnetotactic bacteria are also remarkably regular in size
324 J- L. KIRSCHVINK, F. L. TABRAH AND S. BATKIN
and shape (Balkwill, Maratea & Blakemore, 1980), although a great deal of variatioli
exists between different types (Kirschvink & Gould, 1981). Similarly, the magnetic
properties reported here are consistent with the interpretation that cells of each
tumour type use the same genetic blueprint for constructing their magnetic crystals.
This situation would be difficult or impossible to produce inorganically in nature.
Thermomagnetic, X-ray, Mossbauer, or electron diffraction analyses have identified
magnetite as the source of the ferromagnetism in all organisms and tissues where it
has been extracted, purified or otherwise examined to date (bacteria, Frankel et al.
1979; chitons, Lowenstam, 1962; honey bees, Gould et al. 1978; pigeons, Walcott
et al. 1979; dolphins, Zoeger et al. 1981; tuna fish, Walker & Dizon, 1981; sea turtles,
Perry, Bauer & Dizon, 1981). If magnetite is also the mineral responsible for the
tumour remanence, the average volume present per cell would be 4-5 x io~16 ml in
Lewis lung tumour and 2-5 x io~16 ml in YC-8 lymphoma, corresponding to cubes
with dimensions of 77 and 61 nm respectively. Crystals of this size, if even slightly
elongate in shape, will behave in general as single-domain ferromagnets, a property
which is necessary for and consistent with the presence of a magnetic remanence in
these tissues (Kirschvink & Gould, 1981). Although these data alone cannot deter-
mine the number of discrete crystals within each cell, individual particles less than
about 35 nm in diameter would be superparamagnetic instead of single-domain and
would not hold a stable magnetization. This puts an upper bound of about 11 and 6
crystals per Lewis lung tumour and YC-8 lymphoma cell, respectively.
Several problems arise, however, in the attempt to locate and identify these crystals
using standard techniques on a transmission electron microscope (TEM). Firstly,
there are only a few of these magnetic particles present per cell; consequently their
relative density in the tissue is quite low. A typical 100 nm thick sample mounted on a
TEM grid 1 mm in diameter, for example, would contain a total of only 10-70
crystals. Given their small size, these would be difficult to locate even though they are
naturally electron-dense. The second problem is that other, much more abundant,
subcellular components like pigment granules can have a similar TEM appearance.
Small-area electron diffraction analysis (Towe & Moench, 1981) or X-ray fluorescence
must be performed on each and every candidate particle to establish its crystalline
nature and iron composition. Finally, any acidic step in the sample preparation would
dissolve the small magnetite crystals. These factors probably explain why these
particles have not been recognized in previous TEM studies. A more realistic initial
approach might be to perform subcellular fractionation and centrifugation experi-
ments and then use the magnetic remanence to pinpoint organelles associated
with the ferromagnetic material. The tumour growth effects noted below together
with the observations by Walsh, Shulman & Heidenreich (1961) of ferromagnetic
inclusions in nucleic acid samples suggest that the nucleus would be a good place for
an initial look.
At this stage, the biological function of these magnetic crystals also remains a
mystery. The simplest hypothesis would be that they constitute some form of iron
storage. Many tumours, however, have high internal concentrations of the iron storage
protein ferritin (Dorner et al. 1980) and would not need to use magnetite for this
purpose. Some forms have so much ferritin that radioisotope-labelled antiferritj|
antibodies have been used to locate tumours clinically (Order, Klein & Leichne^
Ferromagnetism in ttvo mouse tumours 325
81). It ia interesting to note that the core of the ferritin macromolecule is made of the
mineral ferrihydrite, the precursor of magnetite in chiton teeth (Kirschvink & Lowen-
stam, 1979). High ferritin levels might lead to magnetite precipitation as a by-product,
but this theory does not explain the apparent crystal size uniformity nor the relatively
small fraction of the cell's total iron content that is present in the ferromagnetic form.
Ferritin is paramagnetic and cannot produce the observed remanence.
Many mouse tumours are known to be induced by viral action, and one possibility
to consider is the production of magnetite under viral control. Although a virus has
apparently not been found in association with Lewis lung tumour, YC-8 lymphoma is
associated with a 45 nm RNA Moloney-sarcoma virus (MSV) in T-lymphocytes.
This virus is probably too small to contain single-domain magnetite particles itself,
but it might cause them to form somewhere else in the cytoplasm. In turn, this would
indicate that the magnetic material is not in the nucleus.
Strong static or weak oscillating magnetic fields have been reported to influence
adversely the growth rate of in vivo tumours (Batkin & Tabrah, 1977; Barnothy, 1964;
Kim, 1976; Weber & Cerilli, 1971; Winterberg, 1967). Previously, these effects have
been interpreted as arising from unspecified paramagnetic interactions or from in-
duced ionic currents or ion motion in the cell cytoplasm. The presence of ferromagne-
tic crystals, however, provides at least two additional interpretations. Firstly, direct
magnetomechanical motion of the crystals produced by their strong (> > kT) inter-
actions with the applied magnetic fields and field gradients may damage adjacent
subcellular organelles and, thereby, inhibit cell growth. Secondly, the relatively high,
metallic-type electrical conductivity of magnetite, roughly io8 to io4 better than
cytoplasm, may yield locally induced current densities that are strong enough to
damage neighbouring structures, depending on the frequency and intensity of the
apphed electromagnetic fields.
Answers to these questions clearly await completion of the micro-anatomical and
mineralogical characterization of the ferromagnetic particles. The discovery of the
presence of ferromagnetic material in the YC-8 lymphoma and Lewis lung tumour
cells led to further studies of the direct effects of magnetic fields on these tumour cells
without the addition of paramagnetic compounds (in preparation).
We thank C. E. Helsley and B. Keating at the Hawaii Institute of Geophysics for
the use of their paleomagnetics laboratory, J. Szekerczes of the Cancer Center of
Hawaii for technical help with the tumour studies, and Roy Yee of Kerns, Inc. of
Honolulu for engineering advice and equipment. This work was supported directly
and indirectly by NSF grants SP179-14845, EAR78-03204, and BNS78-24754
(J. L. K.); and by Cancer Center Support Grant CA-15655 and Leahi Foundation
Grant (S. B. and F. L. T.). Contribution No. 3742 of the Division of Geological and
Planetary Sciences of the California Institute of Technology.
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Ferromagnetism in two mouse tumours

Antiferritin IgG antibody for isotopic cancer therapy.

Carcinoembryonic antigen and ferritin in patients with lung cancer before and during therapy.

Effects of alternating magnetic field on transplanted neuroblastonM Res.

Electron-optical characterization of bacterial magnetite.

Ferritin synthesis by human T lymphocytes.

Ferromagnetic coupling to muscle receptors as a basis for geomagnetic sensitivity in animals.

Ferromagnetic crystals (magnetite?) in human tissue.

Ferromagnetic inclusions in nucleic acid samples.

Identification of magnetite in tuna.

Inhibition of tumour cell growth by the use of non-homogeneous magnetic fields.

Magnetic material in the head of the common Pacific Dolphin.

Magnetite in freshwater magnetotactic bacteria.

Magnetite in the green turtle.

Magnetometers using RF-driven SQUIDS and their application in rock magnetism and paleomagnetism.

Mineralization and magnetization of chiton teeth: Paleomagnetic, sedimentologic and biologic implications of organic magnetite.

Paleomagnetism and Plate Tectonics.

Pigeons have magnets.

Serum ferritin and neuroblastoma.

Serum tumor ferritins in primary liver cancer.

Some possible effects of static magnetic fields on cancer.

Some theoretical considerations on the inhibition of tumor growth by ultrastrong magnetic fields.

Ultrastructure of a magnetotactic spirillum.

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Ferromagnetism in two mouse tumours

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