Toxicology and Applied Pharmacology 199 (2004) 104 – 117

www.elsevier.com/locate/ytaap

Review
Causes of childhood leukaemia and lymphoma
Tracy J. Lightfoot * and Eve Roman
Leukaemia Research Fund Epidemiology and Genetics Unit, Department of Health Sciences, University of York, YO10 5DD, UK
Received 1 October 2003; accepted 8 December 2003
Available online 13 April 2004

Abstract

Childhood cancer is rare comprising less than 1% of all malignancies diagnosed each year in developed countries. Leukaemia is the
commonest form of cancer in children accounting for around a third of all childhood cancer, with acute lymphoblastic leukaemia (ALL) being
the most prevalent. Biologically specific subtypes of ALL and acute myeloblastic leukaemia (AML), the other major morphological type of
childhood leukaemia, are characterised by chromosomal changes. Whilst over 200 genes have been associated with chromosomal
translocations, to date, only MLL, TEL, and AML1 have been linked with childhood leukaemia. Interestingly, there is increasing evidence to
support the theory that gene rearrangements such as these may originate in utero.
As with many other human diseases, both genetic and environmental factors have been implicated in the aetiology of the disease.
Although much has been documented with regard to diet, smoking, alcohol consumption and recreational and prescription drug use during
pregnancy, there is no consistent evidence to support a link with any of these factors and childhood leukaemia. However, findings from
studies investigating prenatal and early life exposures are often based on small numbers of cases as both the type of cancer and exposure are
rare. Furthermore, accurate information relating to past exposures can be difficult to obtain and is often reliant on self-reporting.
To further our understanding of the aetiology of childhood leukaemia and lymphoma, there are areas which clearly warrant investigation.
These include collection of parental dietary folate data combined with genetic analysis of the folate related genes, in utero exposure to DNA
topoisomerase II inhibitors, and the possible effects of assisted reproduction technology on disease susceptibility.
D 2004 Elsevier Inc. All rights reserved.

Keywords: Childhood leukaemia; Lymphoma; Assisted reproductive technology; Folate; Topoisomerase inhibitor

Introduction and three-quarters are male (Parkin et al., 1988; UK Child-

Cancer in children under 15 years old is rare accounting
for less than 1% of all malignancies diagnosed each year in
developed countries (Coleman et al., 1999; Draper, 1995).
Childhood cancers tend to differ from those diagnosed in
adults in terms of their site of occurrence, histological
appearance, and clinical behaviour—growing rapidly, being
aggressively invasive, and being more responsive to che-
motherapy (Miller and Young, 1995). Leukaemias are the
most common cancer to effect children, accounting for
between 25% and 35% of all childhood cancers (Parkin et
al., 1988). A larger number of haematological malignancies
are seen in boys compared to girls, the greatest differences
being observed for lymphomas where between two-thirds
* Corresponding author.
E-mail address: Tracy.Lightfoot@egu.york.ac.uk (T.J. Lightfoot).
hood Cancer Study Investigators, 2000).
The biological heterogeneity of childhood leukaemia is
well documented (Biondi et al., 2000; Kersey, 1997), with
the major morphological types being acute lymphoblastic
leukaemia (ALL) and acute myeloblastic leukaemia
(AML). ALL is the predominant leukaemia seen in child-
hood, with AML accounting for around 15 –17% of cases
in white populations and a slightly higher proportion in
Asia and black populations of North America (Parkin et
al., 1988). Chronic forms of leukaemia in children are very
rare in all populations and seldom exceed 4% of all
leukaemia diagnoses.
Subtypes of AML and ALL are frequently characterised
by genetic alterations, including point mutations and dele-
tions, as well as chromosomal translocations, including
chimeric or fusion genes and changes in chromosome
number (hyperdiploidy or hypodiploidy) (Look, 1997; Rai-
mondi, 1999). Although over 200 genes have been found to

0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2003.12.032
 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117 105

be involved in chromosomal translocations, many of them Mechanisms of exposure

are rare and only certain genes have been associated with
childhood leukaemia (Table 1) (Greaves and Wiemels,
2003). These include MLL, TEL, and AML1, all of which
can fuse with over 15 other genes and in the case of TEL and
AML1 with each other (Golub et al., 1995; Romana et al.,
1995).
In developed countries, the incidence of leukaemia rises
rapidly after birth peaking at around 3 years of age before
declining and then rising steadily again throughout life
(Linet and Devesa, 1991). The different subtypes of ALL
tend to segregate by age, which may account for the marked
prognostic differences among infants, children, and adults
(Greaves, 1999). In infants less than 12 months old, a
distinct type of ALL with a pro-B phenotype and chromo-
some rearrangements in the MLL gene has been identified.
The marked peak in incidence between 2 and 5 years of age,
associated with common B-cell ALL, has resulted in many
aetiological hypotheses, most notably those involving ex-
posure to infections (Greaves and Alexander, 1993; Kinlen,
1995; Law et al., 2003).
As with most other cancers, the mechanism by which
leukaemia arises is likely to involve gene – environment
interactions—the environmental exposures being derived
from both endogenous and exogenous sources. According-
ly, it is important to identify exposures that cause DNA
damage and induce chromosome breaks which are inade-
quately repaired, ultimately leading to disease initiation and
progression.
In adults, carcinogens can accumulate over a long
period of time, whereas in children, the potential exposure
period is much shorter. Accordingly, exposures acting
before birth and early in life have long been thought to
be important determinants of leukaemia, and the list of
suspected chemical, physical, and biological agents con-
tinues to increase. Unfortunately, the evidence regarding
the majority of suggested exposures is limited and often
contradictory.
Several potential mechanisms by which exogenous
agents including environmental factors and drugs could
impact on childhood cancer susceptibility have been iden-
tified (Fig. 1). Early in a female’s life, contact with putative
carcinogenic or toxic substances may play a role since
potentially toxic agents or their metabolites could cause
permanent damage. Indeed, it could be argued that, since no
new oocytes are formed after birth and their maturation
commences during gestation, exposures acting within this
critical window may be the most important. Accordingly,
germ cell mutations arising in the offspring (index child)
could be attributable to exposures acting on the maternal
grandmother whilst she was pregnant with the index child’s
mother. Conversely, paternal exposures may play a more
direct role as spermatogenesis continues from puberty to old
age and therefore there is a greater opportunity for the
accumulation of gene mutations in men (Cavalli-Sforza
and Bodmer, 1971).

Table 1
The major biological subtypes of childhood leukaemia and associated chromosomal changes
Subtype Cell type involved Chromosome Molecular lesion Frequency (%) Functional product
abnormality
Acute B-cell 11q23 MLL-AF4, MLL-ENL, approximately 85 modified transcription
lymphoblastic progenitor-monocytica translocations and other fusions (of infant ALL) factorb
leukaemia (infants) approximately 5
(ALL) (of total ALL)
B-cell precursor hyperdiploidy increased gene dosage approximately 35 unknown
(of B-cell precursor ALL)
t(12;21)(p13;q22) TEL-AML1 fusion approximately 20 chimeric transcription
(of B-cell precursor ALL) factorc
t(1;19)(q23;p13) E2A-PBX1 fusion approximately 5 chimeric transcription
(of B-cell precursor ALL) factor
t(9;22)(q34;q11) BCR-ABL fusion approximately 5 activated kinase
(of B-cell precursor ALL)
T-cell precursor 1q deletion; SIL-SCL fusion approximately 25 dysregulated
t(1;14)(p32;q11) (of T-cell precursor ALL) transcription factor
(SCL/TAL1)
Acute myeloid In infants 11q23 MLL-AF6, -AF9, -AF10, approximately 50 modified transcription
leukaemia translocations or other fusions (of infant AML) factorb
(AML)
t(8;21)(q22;q22) AML-ETO fusion approximately 15 chimeric transcription
(of total AML) factorc
Adapted from Greaves and Wiemels (2003).
a
This subtype has phenotypic features of both early B-lineage progenitors and monocytes.
b
MLL may modify chromatin structure and control the expression of key developmental genes (such as HOX).
c
AML-1, which is an important positive transcriptional factor, may be switched to a transcriptional repressor in these fusions (Tenen, 2003; Speck and
Gilliland, 2002).
106 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117
Fig. 1. Possible exposure pathways for cancer aetiology.
Exposures acting during pregnancy can either do so
directly (e.g. ionising radiation) or indirectly via the
placenta (e.g. maternal drug ingestion)—the effect that
the latter may have being dependant not only on timing
and dose but also on the mother’s, as well as the
embryo’s, ability to metabolize the agents involved.
Interestingly, not only is cytochrome P450 2D6, which
is responsible for the metabolism of over 40 clinically
used drugs including beta-blockers and analgesics, poly-
morphic (10% of the Caucasian’s are poor metabolisers)
but its activity is induced during pregnancy. With respect
to in utero exposures, it is also important to remember
that maternal preconceptional contact with agents that
have a slow metabolic clearance may result in the mother
retaining embryotoxic doses into the early stages of
pregnancy.
After birth, as well as being influenced by direct envi-
ronmental exposures, the infant can still be affected by
parental exposures, which for breast-fed infants may have
occurred many years in the past. Clearly, the ability of
offspring to detoxify and excrete carcinogens to which they
are exposed is important.
Exposures
Whilst animal models have shown that preconceptional
exposure of germ cells to carcinogens and mutagens results
in excess tumours in offspring, no confirmed preconcep-
tional exposure links to cancer in humans have been
demonstrated. There is, however, evidence to suggest that
human fetal exposure to potentially toxic or carcinogenic
substances can increase cancer risk in children and young
adults. Nonetheless, although several factors have been
suggested, including infections, folate, lifestyle, and drugs,
the only accepted human in utero carcinogenic exposures
are ionising radiation (Stewart et al., 1956, 1958) and
diethylstilbestrol (Herbst and Ulfelder, 1971).
Lifestyle
General lifestyle factors form the broad basis for many
maternal exposures, including diet, smoking, alcohol, and
recreational drug use in addition to vitamin supplementa-
tion, prescription medications, and exposure to infections.
Whilst the range of relevant paternal exposures during
pregnancy is clearly more limited, it is important to remem-
ber that paternally mediated effects via intercourse and
general environmental exposures (e.g. smoking) may be
important.
Cigarette and alcohol consumption
Adverse health effects of cigarette smoking and exces-
sive alcohol consumption are well documented—both with
respect to the health of the individual (Doll, 1998; Rehm et
al., 2003) and with respect to the health of their offspring
(Kallen, 2001). However, although many studies have
reported on this topic, no consistent evidence to confirm
links with childhood leukaemia/lymphoma has been docu-
mented. Although Sorahan et al. (1995, 1997), in their
 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117
analysis of data from the Oxford Survey of Childhood
Cancers, reported a weak association between maternal
smoking during pregnancy and childhood ALL, and a
significant trend between increasing paternal tobacco con-
sumption and childhood cancer, these observations have not
been confirmed in other studies. Recently, confirming
earlier findings for leukaemia from the Children’s Cancer
Group (Brondum et al., 1999), data from the UKCCS failed
to provide any evidence that parental smoking is a risk
factor for leukaemia or any other form of childhood cancer
(Pang et al., 2003).
There is less information about the relationship be-
tween parental alcohol intake and childhood leukaemia,
and the majority of studies have focussed primarily on
maternal consumption during pregnancy. Whilst no con-
sistent association between maternal alcohol intake and
ALL, or all leukaemias combined, have emerged, positive
associations have been reported for AML (Severson et al.,
1993; Shu et al., 1996; van Duijn et al., 1994). However,
there were differences in the findings of these studies;
Severson et al. (1993) reported that the risk was more
pronounced when the disease had a monocytic component
(M4/M5), while Shu et al. (1996) reported that the risk
was higher for the subgroup with the M2/M2 morphology.
As far as we are aware, no convincing associations for
paternal alcohol intake and risk of childhood leukaemia
have been reported (Severson et al., 1993; Shu et al.,
1988, 1996).
Parental drug use
Parental drug use has been of particular interest ever
since Herbst and Ulfelder (1971) reported an association
between maternal use of diethylstilbestrol in pregnancy
and clear cell adenocarcinoma of the vagina in young
women. Further maternal use of certain drugs has been
associated with a range of adverse reproductive outcomes
including fetal death, low birth weight, congenital abnor-
mality, and physical and developmental delays in offspring
(Bennett, 1999; Broekhuizen et al., 1992; Czeizel et al.,
1999; D’Apolito, 1998; Khera, 1987; Myrianthopoulos
and Melnick, 1987; Richardson et al., 1996). Accordingly,
there is continuing concern about the impact of many of
these drugs on childhood cancer, particularly leukaemia,
especially since many prescription drugs are used more
frequently during pregnancy (Collaborative Group on
Drug Use in Pregnancy (C.G.D.U.P), 1992). It has been
reported that up to 90% of women take three to four
different drugs during the antenatal period (Collaborative
Group on Drug Use in Pregnancy (C.G.D.U.P), 1992;
Walker, 1999), with nutritional supplements, such as iron,
folate, calcium, and vitamins, being the most commonly
prescribed (Collaborative Group on Drug Use in Pregnan-
cy (C.G.D.U.P), 1992; Das et al., 2003). Additional
classes of drugs taken during pregnancy are to treat
nausea and vomiting, dyspepsia, and other related con-
107
ditions, and include analgesics, spasmolytics, antacids, and
systemic antibiotics (Das et al., 2003; Fonseca et al.,
2002).
Whilst it is feasible to suggest that in utero exposure to
certain medications may have a role in the aetiology of
childhood cancer, there are limited data to support such an
association. Maternal use of marijuana and antiemetic
medication was reported to be linked with childhood
AML (Robison et al., 1989), and in adults, links between
self-prescribed medication and non-Hodgkin’s lymphoma
and leukaemia (Doody et al., 1996) were observed but the
findings were not consistent. More recently, Wen et al.
(2002) reported strong associations between childhood
ALL and maternal use of antihistamines and allergy
remedies.
Data from animal experiments have shown that marijua-
na smoke is both mutagenic and toxic to the fetus (Nahas
and Latour, 1992). In humans, both maternal and paternal
use of mind-altering drugs, of which marijuana was the most
commonly used, was reported to influence the risk of ALL,
with the risk increasing further when both parents used the
drug (Wen et al., 2002). It must be noted that the informa-
tion obtained regarding medication in the study relied on
self-reporting, and in some instances, surrogate reporting by
mothers on behalf of fathers.
DNA topoisomerase inhibitors
The breakage and recombination of the MLL gene with
one of several potential partner genes is a common feature
of infant acute leukaemia with up to 80% having chromo-
somal translocations involving the MLL gene at 11q23.
(Ross et al., 1994). Ford et al. (1993) provided molecular
evidence that rearrangements of the gene at 11q23 could
originate in utero, and in a later report proposed that T-
lineage malignancies in older children may also be initiated
in utero (Ford et al., 1997). Neonatal blood spots from
effected children were used to confirm that this genetic
lesion occurred in utero (Gale et al., 1997), and it has been
suggested that a MLL fusion in the appropriate haemato-
poietic stem cell may be sufficient to cause leukaemia
(Greaves, 1999). Further, since MLL rearrangements are
frequently seen in adult cases of leukaemia following
chemotherapy treatment with topoisomerase II inhibitors,
it is possible that in utero exposure to topoisomerase
inhibitors could also be important in infant leukaemia risk
(Greaves, 1999).
Wen et al. (2002) observed an increased risk of ALL
(although not statistically significant) with maternal use of
laxatives, some of which are topoisomerase inhibitors.
Whilst Alexander et al. (2001) observed elevated odds
ratios for MLL leukaemias following exposure to DNA
damaging drugs, dipyrone and mosquitocidals, it is not
clear whether the results were a consequence of topoiso-
merase II inhibition or affects on other pathways. Howev-
er, the data do suggest that exposures to certain chemicals
108 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117

in utero may result in MLL gene fusions. A recent case which accounts for the peak in incidence in childhood
report (Borkhardt et al., 2003) described a case of con- leukaemia between the ages of 2 and 5 years, was a
genital leukaemia in the offspring of a mother who used consequence of two spontaneous mutations (Fig. 2). It
permethrin, a widely used household pesticide, excessively was suggested that, since in the early stages of development
during pregnancy. Cytogenetic analysis of the child’s bone and infancy, lymphoid precursor cells are more likely to
marrow showed the presence of the 11q23/MLL rearrange- undergo spontaneous mutation than other somatic cells due
ment and further studies confirmed the ability of permeth- to their high proliferative rate and their developmentally
rin to cause the MLL rearrangement in vitro. Although an regulated intrinsic mutagenic activity (Greaves, 1988), then
isolated case is not sufficient evidence on which to base the first mutation is likely to occur in utero during the rapid
definitive conclusions, the data suggest that in utero multiplication of the B-cell precursors in the liver and bone
exposure to permethrin may cause severe side effects in marrow. The second mutation was postulated to occur
fetal haemopoietic precursor cells. postnatally during the proliferation of antibody-producing
The list of topoisomerase inhibitors is, however, exten- cells after the infant’s first exposure to a diverse range of
sive and includes not only chemotherapeutic agents but also antigens. Greaves also proposed that the immune response
benzene metabolites (derived from cigarette smoke and was promoted as a consequence of the indirect up-regulation
pollution for instance), bioflavonoids, herbal medicines, of the number of target B-cells at risk.
anthraquinone laxatives, podophyllin resin, quinolone anti- The response to infection and therefore the likelihood of
biotics, pesticides, and most phenolic chemicals and their developing leukaemia may be influenced by other factors
metabolites. Furthermore, topoisomerase inhibitors have including genetic background, socioeconomic status, vacci-
been found in specific fruits, tea, coffee, wine, soy, and nations, and length of breast-feeding as they can affect the
cocoa and it is quite likely that many other chemicals that
have the ability to inhibit this enzyme have yet to be
identified. Limited data on the topic are available, but one
US case-control study linked childhood leukaemia with
maternal dietary intake of topoisomerase inhibitors (Ross
et al., 1996).
DNA topoisomerase inhibitors are also used to treat HIV
and certain sexually transmitted diseases which have been
shown to augment the predisposition of individuals to HIV.
Whether this can affect the health of offspring is unknown,
and studies are needed to look at disease profiles in children
with HIV-positive parents who were treated with this type of
drug either before conception (father, mother, or both) or
during pregnancy (mother).

Infections

There is no dispute about the fact that certain infectious/

parasitic agents are major causes of some types of cancer,
for example, HTLV-1 in T-cell leukaemia/lymphoma and
Epstein– Barr virus (EBV) in Burkitts lymphoma (Newton
et al., 1999). In addition to these associations with chronic
infection, Greaves (1988, 1997) and Kinlen (1988, 1995)
have suggested that infectious agents may be involved in the
aetiology of childhood leukaemia.
The excess of leukaemia in young people in the locality
of two nuclear reprocessing plants in the United Kingdom
was suggested by Kinlen (1988) to be due to the introduc-
tion of a viral exposure into regions that were previously
predominantly rural and sparsely populated. He argued that
such working environments would have allowed the trans-
mission of microorganisms and the spread of infection
would have been enhanced by population mixing by bring- Fig. 2. Proposed immunological model for the aetiology of childhood
ing together both infected and susceptible individuals (Kin- common acute lymphoblastic leukaemia. Adapted from Greaves (1997) and
len, 1995). In 1988, Greaves proposed that common ALL, taken from Anderson et al. (2000).
 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117
timing and magnitude of positive feedback stimulation to
the lymphoid precursor population in the bone marrow.
Several studies have also examined the relationship between
maternal infection during pregnancy and the subsequent
occurrence of cancer in the offspring although the data are
inconsistent (Adelstein and Donovan, 1972; Doll, 1973;
Fedrick and Alberman, 1972; Fine et al., 1985; Gilman et
al., 1988; Hakulinen et al., 1973; Leck and Steward, 1972;
McKinney et al., 1985; Ross et al., 1994; Stewart et al.,
1958). Infections studied included varicella and influenza,
and it was also suggested that an infection with similar
properties to rubella might be important. Of the three cohort
studies where data are available, Fine et al. (1985) found no
association with maternal infection and childhood leukae-
mia or lymphoma; Vianna and Polan (1976) identified three
cases of lymphatic leukaemia in 63 pregnancies complicated
by varicella; and Fedrick and Alberman (1972) found that
out of 1959 reported maternal exposures to influenza, six
offspring developed ALL and one had Hodgkin’s disease.
Data from the case-control studies show no consistent
associations with influenza or varicella infection during
pregnancy and leukaemia. Lehtinen et al. (2003) investigat-
ed the relationship between maternal herpesvirus infections
and risk of childhood ALL and found that maternal EBV
infection, defined by the presence of specific EBV IgM
antibodies in the serum at 12– 14 weeks gestation, was
significantly associated with ALL development in the off-
spring. They postulated that in utero EBV infection could
result in the genotoxic damage that is responsible for the
MLL and TEL-AML1 gene fusions found in infant leukaemia
and common ALL (Greaves, 1997; Wiemels et al., 1999).
Thus, whilst there is an increasing amount of data accruing
in this area as yet no virus-dependent immunological
mechanisms have been positively identified.
Ionising radiation
Epidemiological evidence that prenatal exposures were
involved in the development of childhood malignancy was
first provided by the Oxford Survey of Childhood Cancers
over 40 years ago, when an association between diagnostic
radiography of mothers during pregnancy was related to the
subsequent development of leukaemia and other cancers in
their children (Stewart et al., 1956, 1958). Although this
association was initially greeted with some scepticism, it is
now generally accepted that the fetus and young child may
be more susceptible to the affects of ionising radiation than
the adult, with modern concern revolving mainly around the
importance of dose and gestational age at the time of
exposure (Mole, 1990; Wakeford, 1995).
It is well established that exposure to high doses of
radiation prenatally and early in life increases cancer rates
in humans and animals (for review, see Anderson et al.,
2000). The suggestion that germ cell damage caused by
low doses of external ionising radiation might increase the
109
risk of cancer and other genetic diseases in the progeny of
those exposed is, however, a controversial topic. The
findings for paternal occupational exposure are one of
the few areas where good data exist. In 1990, Gardner
and colleagues reported that the recorded external dose of
whole body ionising radiation to fathers during their time
at Sellafield was associated with the development of
leukaemia in their offspring. Their results suggested that
the highest risks were seen in those with highest accumu-
lated ionising radiation doses before conception, either
over their total duration of exposure or during the pro-
ceeding 6 months. The offspring of those fathers that had a
lifetime cumulative dose of 100 mSv or more before
conception were estimated to be 8.4 [95% confidence
interval (CI) 1.4 – 52.0] times more likely to develop
leukaemia. In those fathers with a cumulative preconcep-
tional dose of z 100 mSv but who received 10 mSv of
this dose 6 months before their child’s conception, a
relative risk of 6.8 (CI 1.5 – 31.9) was observed. Case-
control studies carried out in the vicinity of other nuclear
establishments where workers were, on average, exposed
to much lower doses were unable to confirm these findings
(McLaughlin et al., 1993; Roman et al., 1993; Urquhart et
al., 1991), and the results of other large-scale studies are
contradictory (Draper et al., 1997; Roman et al., 1999).
In terms of documented human exposures, the Sellafield
situation that Gardner and colleagues were asked to inves-
tigate was unusual in many ways, not the least of which was
the fact that comparable data from other exposed workforces
were not available. Indeed, the paucity of historical human
data on this topic, coupled with the fact that workers
nowadays are exposed to much lower doses, means that
additional human data on this topic are unlikely to be
reported (Anderson et al., 2000).
Nonionising radiation
Wertheimer and Leeper (1979) published their first study
suggesting an association between residential exposures to
extremely low-frequency magnetic fields (ELF-EMF) and
cancer in children around 25 years ago. Ever since the
potential aetiological impact of these fields on childhood
cancer in general—and leukaemia in particular—has been
the subject of considerable public concern and scientific
interest.
The last decades have seen a massive increase in man-
made sources in the power generation part of the electro-
magnetic spectrum, with man-made fields now being many
thousands of times greater than their naturally occurring
counterparts. This change is undoubtedly one of the reasons
behind people’s concerns over the possible adverse health
effects of these exposures. Electrical energy involves both
voltage and current. Electric fields occur wherever there is a
voltage; for example, when the flex of a lamp is connected
to a main plug which is live, electric fields are produced
110 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117

even when the lamp itself is switched off. By contrast, Folate

magnetic fields are only present when current is actually
flowing. Magnetic fields, produced when the lamp is
switched on and current flows, are difficult to screen and
will pass readily through the fabric of a building, but trees
and building materials for example will screen electric
fields.
To date, experiments on animals and isolated cells have
failed to support the suggestion that exposure to power
frequency fields may be carcinogenic, and an individual
record-based pooled analysis of information on over 3000
cases of cancer and 10 000 controls from Europe and North
America has reported that for magnetic field exposures up to
0.4 AT, the data demonstrated relative risks near the no-
effect level (Ahlbom et al., 2000). For the very small
proportion (0.8%) of subjects with exposure above 0.4 AT,
the data showed a 2-fold increase, which the authors
concluded was unlikely to be due to random variability
but could have been due to selection bias (Ahlbom et al.,
2000).
Information on electric field exposure is much sparser
than that on magnetic fields, but a recently published UK
study on this topic—which contains a thorough review—
found no support for the hypothesis that residential
exposures to ELF electric fields were associated with
childhood cancer (Skinner et al., 2002). Furthermore, the
authors concluded that electric field exposure could
certainly be excluded as a cause of a substantial propor-
tion of leukaemia or other childhood malignancies in the
United Kingdom.
In summary, once the spectre of risk has been raised,
proving a negative is virtually impossible. However, there
is now a large body of evidence suggesting that expo-
sure to generally encountered power frequency magnetic
and electric fields are not associated with the develop-
ment of childhood cancer in general or with leukaemia
in particular.
Dietary folate is derived from fruit and vegetables, and
high-folate intake has previously been associated with
decreased risk of colorectal adenoma and carcinoma along
with cervical dysplasia in adults (Kim, 1999). The associ-
ation between folate deficiency and cancer risk is thought
to be due to altered DNA methylation and impairment of
DNA synthesis and repair (Fig. 3). DNA methylation is
compromised due to diminished levels of available S-
adenosylmethionine, the principal donor for DNA meth-
ylation. Inadequate levels of folate cause a reduction in
the availability of 5-methylenetetrahydrofolate for the
methylation of homocysteine to methionine and subse-
quently the DNA methylation donor. Folate, in the form
of 5,10-methylenetetrahydrofolate (5,10-MTHFR), acts as
a methyl donor for the conversion of deoxyuridylate to
thymidylate. Consequently, folate deficiency impairs this
process, potentially causing incorporation of uridine into
DNA instead of thymidine. Chromosomal aberrations and
malignant transformations may occur following destabili-
sation of the DNA and subsequent strand breaks as a
result of the repeated attempts by repair enzymes to
excise the uracil.
Methylenetetrahydrofolate reductase (MTHFR) is re-
sponsible for the conversion of 5,10 methylenetetrahydro-
folate to 5-methyltetrahydrofolate (Fig. 3) and hence is
important in regulating DNA methylation. Common poly-
morphisms in the MTHFR gene have been reported at
positions 677 and 1298 resulting in alanine-to-valine and
glutamate-to-alanine substitutions, respectively (de Fran-
chis et al., 1995; Frosst et al., 1995; Nishio et al., 1996;
Van der Put et al., 1998; Weisberg et al., 1999). Both of
these polymorphisms result in diminished enzyme activity.
Up to 15% of the population have been identified as
homozygotes for the 677 allelic variant with allele fre-
quencies of up to 33% reported for the 1298 polymor-

Fig. 3. Overview of the human folate metabolic pathway. SAM—adenosylmethionine; SAH—S-adenosylhomocysteine; DHF—dihydrofolate; DHFR—
dihydrofolate reductase; THF—tetrahydrofolate; SHMT—serine hydroxymethyltransferase; 5,10-methyleneTHF—5,10-methylenetetrahydrofolate; MTHFR—
5,10-methylenetetrahydrofolate reductase; 5-methylTHF—5-methylenetetrahydrofolate; 10-formylTHF—10-formyltetrahydrofolate; MS—methionine syn-
thase; dTMP—deoxythymidine monophosphate; dump—deoxyuridine monophosphate.
 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117
phism (Frosst et al., 1995; Van der Put et al., 1998;
Weisberg et al., 1999).
Following studies examining the affects of polymor-
phisms of the MTHFR gene on cancer risk, there is further
evidence to support the role of folate in human carcinogen-
esis and importantly in susceptibility to ALL. Skibola et al.
(1999) demonstrated that polymorphisms in the MTHFR
gene (C677T and A1298C) are associated with reduced risk
of ALL in adults. Similar findings have been reported for
children in the UK Childhood Cancer Study where a
protective effect of the C677T polymorphism in infant
leukaemia with MLL rearrangements was observed (Wie-
mels et al., 2001). Franco et al. (2001) also demonstrated a
protective effect of the C677T polymorphism in the MTHFR
gene in infant leukaemia with MLL rearrangements and in
hyperdiploid leukaemia but found that the A1298C poly-
morphism did not significantly affect the risk of ALL in the
population studied. In contrast, a more recent study by Balta
et al. (2003) in Turkish children reported that the MTHFR
C677T polymorphism did not significantly affect the risk of
developing childhood ALL.
Examination of other genes involved in folate metabo-
lism suggests that variations in other aspects of its uptake
and metabolism may be important for adult ALL (Skibola et
al., 2002). The folate metabolising genes found to play a
role in adult ALL include thymidylate synthase (TS) and
serine hydroxymethyltransferase (SHMT), both of which
are involved in DNA synthesis. This suggests that uracil
mis-incorporation and resultant chromosomal aberrations
may be important in the pathogenesis of ALL. These
findings warrant further investigation and future studies
should include genotyping of children and their parents
for polymorphisms in genes involved in the metabolism
and transport of folate.
It is also possible that the affects of the polymorphisms in
MTHFR and related genes on cancer risk will be dependant
on folate status. Indeed, a strong protective effect of folate
supplementation ( F iron) in pregnancy on the risk of
common ALL in the child has been reported. However,
self-reported iron supplementation was only weakly protec-
tive, indicating that if there is a role for folate or iron that it
is predominantly the folate that has an effect or the folate in
combination with the iron rather than iron alone (Thompson
et al., 2001). The time during pregnancy when the folate
was taken or the duration of exposure did not influence the
strength of the association observed.
For example, in the presence of adequate amounts of
folate, decreased MTHFR activity could lead to an in-
crease in 5,10-methylenetetrahydrofolate resulting in less
mis-incorporation of uracil into DNA and hence less
double-strand breaks. Conversely, if folate levels are inad-
equate, the DNA-damaging effects of hypomethylation
may outweigh the beneficial effects of an increase in the
levels of 5,10-methylenetetrahydrofolate (Thompson et al.,
2001). This merits the collection of detailed good quality
information on maternal folate intake in combination with
111
genetic analysis of the folate-related genes in future
studies.
Infant feeding
Much has been documented about the potentially pro-
tective affects of breast-feeding on childhood leukaemia
risk since breast-feeding has been shown to provide many
immunological benefits to the offspring (Cutherbertson,
1999; Goldman, 1993; Jelliffe and Jelliffe, 1978; WHO
Collaborative Study Team on the Role of Breast Feeding
on the Prevention of Infant Mortality, 2000). Shortly after
birth, breast milk contains antimicrobial immunoglobins
and later on lymphocytes, macrophages, and soluble fac-
tors that may contribute to an altered response to infection
(Hanson, 2000). Conversely, breast-feeding also allows
transfer of various infectious agents from the mother to
the child (Nduati et al., 2000; Ruff, 1994).
Greaves’ delayed exposure hypothesis predicts a protec-
tive effect for protracted breast feeding, specifically for
childhood ALL as a result of priming the developing
immune system by transmission of both infective agents
and immunological properties of breast milk (Greaves,
1997). Whilst many studies have shown strong evidence
for a link (Bener et al., 2001; Perrillat et al., 2002; Schüz et
al., 1999; Shu et al., 1999), the UK Childhood Cancer Study
reported only weak evidence of a protective effect of breast-
feeding and only with regard to overall childhood cancer
incidence (Beral et al., 2001). More recently, data from the
Oxford Survey of Childhood Cancers also failed to support
the hypothesis, as there was no evidence of protection from
breast-feeding for either ALL or for all cancers combined
(Lancashire and Sorahan, 2003).
The deleterious effects of breast-feeding have not been
as extensively studied but involve the possibility that
mutagenic and carcinogenic compounds along with viruses
such as hepatitis-B virus (HBV) and human immunodefi-
ciency (HIV) can be transferred to the offspring. It must be
noted that the maternal exposure to any of these agents
(viruses, carcinogens, mutagens) may have occurred several
years before the pregnancy. Phillips et al. (2002) showed
that human breast milk contains chemicals that are capable
of inducing genotoxic damage in test systems and in human
epithelial cells. What is of interest is that the genotoxic
activity seen with the breast milk was not due to tobacco
smoke and further work is underway to identify what
chemicals are inducing the genotoxic response. In addition,
Phillips et al. (2002) are currently conducting a small-scale
study involving 20 volunteers each keeping a diet diary for
28 days and giving a daily breast milk sample to see how
the effect of diet may influence the composition of breast
milk and its genotoxic activity. The exact effect of the
transmission of genotoxic substances via breast milk on the
offspring is not clear but is an area that warrants further
investigation.
112 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117
Fertility
Maternal fertility is a subject of much interest with regard
to investigating origins of childhood disease but there are
limited epidemiological studies in this area. Almost 20 years
ago, Van Steensel-Moll et al. (1985) observed positive
associations between childhood ALL and use of drugs to
maintain pregnancy, hospitalisation and/or consultation for
subfertility and a history of two or more miscarriages
implying that impaired maternal fertility may be involved
in the aetiology of childhood leukaemia. However, data in
this area are sparse and often contradictory (Cnattingius et
al., 1995; Kaye et al., 1991; MacMahon and Newill, 1962;
Shu et al., 1988). Ross et al. (1997) observed a positive
association between leukaemia diagnosed before the first
birthday and number of fetal losses in the AML subgroup.
More recently, it has been claimed that induced abortion
prior to pregnancy and oral contraceptive use during preg-
nancy are associated with risk of ALL from all immuno-
phenotypic subtypes combined (Ou et al., 2002).
Twenty-five years on from the birth of the first ‘‘test-tube’’
baby, there are still limited data available to look at the
adverse health outcomes in the offspring resulting from
assisted reproductive technologies (ART). It is known that
multiple pregnancies are a common feature of ART, which is
an important factor in the assessment of the health of the
offspring at birth (Beral and Doyle, 1990; Doyle, 1996,
1999), but little has been reported with regard to prenatal
exposure to high levels of oestrogen and related drugs used to
induce super-ovulation. Possible increases in cancer inci-
dence in children born as a result of in vitro fertilisation have
been observed in two case reports (Kobayashi et al., 1990;
White et al., 1990), which has highlighted concerns over the
prenatal exposure (before and after conception) to high levels
of oestrogen and related compounds used for ovarian stim-
ulation. A study of UK births following ART did not show an
excess incidence of childhood cancer, although, as the
authors noted, the study population was probably too small
to detect an increase in incidence, even if one existed (Doyle
et al., 1998). Public interest in this area is at an all time high
following recent comments in the popular press and also in
light of discussions in the UK that IVF treatment is to be
made available on the National Health Service. Thus, there is
a need to delineate potential effects of ART on disease
susceptibility in children born as a result of the technology.
Summary/Discussion
Genetic and environmental factors have been implicated
in the aetiology of many human diseases including cancer in
both adults and children. However, data to support such
hypotheses are often inconsistent and, in some cases,
contradictory. Epidemiological studies can provide a wide
range of relevant data; for instance, diagnostic information
and medical histories provide good quality information, and
if biological samples are available, then it is possible to
obtain diverse and comprehensive genetic information. On
the other hand, with respect to environmental exposures
(e.g. diet, smoking, alcohol), epidemiological studies fre-
quently rely on self-reported data which can be problematic.
Even when self-reported exposure data are sound, accurate-
ly quantifying the dose, the precise time, and the length of
exposure is seldom clear-cut. Moreover, individual expo-
sures and the timing of exposures cannot always be readily
isolated from one another, and so observed associations with
one factor may simply reflect associations with others.
Furthermore, studies investigating prenatal and early life
exposures are often limited to small numbers of cases since
the type of cancer and indeed many of the putative delete-
rious exposures are rare.
Although many possible environmental agents have been
suggested as risk factors for childhood leukaemia and
lymphoma, much of the evidence is conflicting and contra-
dictory and the search for candidate exposures—assuming
they exist—continues. With respect to genetic analyses,
there is evidence to suggest that gene rearrangements can
originate in utero (Ford et al., 1997; Wiemels et al., 1999,
2001). However, whilst most research conducted to date has
concentrated on examining index children, it is important to
remember that further insights may be gained by examining
other family members (Christensen et al., 1999; Labuda et
al., 2002).
Questions and Answers
You have reviewed positive associations between maternal
alcohol intake and acute myeloblastic leukemia (AML),
citing three studies
Q: What was the magnitude of the increased risk?
A: Severson et al. (1993) reported an increased risk (odds
ratio = 3.0, 95% CI 1.2 – 8.4) for AML diagnosed at or
before the age of two, however, when the disease had a
monocytic component the risk increased to 9.0 (95% CI,
1.3– 394.5, based on 12 discordant pairs). In the study of
van Duijn et al. (1994), the risk for reported consumption of
alcohol during pregnancy was 2.6 (95% CI, 1.4 – 4.6),
however, this was only apparent in the 0 –4 and 5 – 9 year
subgroups and not in the 10– 14 age group. Shu et al. (1996)
also reported a raised risk of 2.6 (95% CI, 1.1– 5.0) for
maternal alcohol consumption during pregnancy and risk of
AML. A dose response relationship was also observed in
this study. The odds ratio for 1– 20 drinks during pregnancy
compared to none was 2.4 (95% CI, 1.1– 5.0), this increased
to 3.1 (95% CI, 1.2 – 8.1) when the number of drinks
consumed during pregnancy was more than 20.
Q: Have there been other studies that have not seen this
association?
A: As far as we know, the only studies that have reported
any association, either positive or negative, with maternal
 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117
alcohol intake and AML are the three that are cited in the
manuscript.
Q: Is there a biologically plausible mechanism to ac-
count for such a cell-specific effect of alcohol?
A: As far as we are as aware to date there is no
mechanism to account for the cell specific effect of alcohol.
It must be noted that data is collected on maternal alcohol
intake because in simplistic terms it can be!
You refer to a report of strong associations between
childhood acute lymphoblastic leukemia (ALL) and maternal
use of certain drugs, including immune system targeting
agents such as anti-histamines and allergy remedies
Q: What was the magnitude of increased risk?
A: It is important to note that data relating to the use
of medications during pregnancy are often self-reported.
Wen et al. (2002) reported that the maternal use of
antihistamines or allergic remedies produced an odds ratio
of 4.3 (CI 99%, 0.6 –32.1) in children diagnosed under
the age of 1 with ALL. In addition, an association with
ALL was also reported (although not significant) between
maternal laxative use, some of which are topoisomerase
inhibitors.
Q: Are any of these known to be DNA damaging and/or
topoisomerase inhibitors?
A: As far as the authors are aware, to date, neither
antihistamines nor allergic remedies have been classified
as topoisomerase inhibitors, or as known DNA damaging
agents. However, as documented in the manuscript, the list
of known topoisomerase inhibitors is not complete and it is
quite likely that many other drugs will have the capacity to
inhibit the enzyme.
Q: Could these agents alter immune system development
in the fetus?
A: Possibly-maybe this is an area that warrants further
investigation.
Q: Further, could the real culprit here be, not the drugs
themselves, but the underlying immunological phenomena
which they were taken to treat, such as a genetic profile or
an antigen exposure history that could predispose to ALL
development?
A: Separating the effects of drugs from the diseases they
were given to treat is difficult. Clearly, this is an interesting
area that requires a more detailed investigation than has
hitherto been undertaken.
The new report of association of maternal EBV infection
with offspring ALL is intriguing. EBV effects in adults have
probably been well studied
Q: Is this virus known to cause any of the types of DNA
damage characteristically seen in childhood ALL?
A: The majority of studies on this topic have been based
on small numbers of cases. There have been previous
studies investigating possible associations between maternal
113
EBV infection and childhood leukaemia and lymphoma.
Ambinder et al. (1993) reported that EBV contributed to the
pathogenesis of Hodgkin’s disease and Schlehofer et al.
(1996) reported that antibodies to EBV were frequently
found in children with leukaemia diagnosed prior to the age
of six and concluded that their data suggested that EBV may
play a role in the development of childhood leukaemia. The
Epstein-Barr virus (EBV) infects and immortalizes B cells
resulting in polyclonal proliferation which would normally
be abrogated by EBV-specific cytotoxic T cells (CTL).
However, in immunosuppressed subjects this mechanism
is inadequate and the B-cell proliferation goes unchecked
and may evolve into an oligoclonal or monoclonal lympho-
proliferative disorder. Recently, Corapcioglu et al. (2003)
reported a MLL-AF4 gene rearrangement in a child with
Epstein-Barr virus-related post-transplant B-cell lymphoma,
and Fasan et al. (2003) also reported Epstein-Barr virus-
related post-transplant lymphoproliferative disorder with
t(9;14)(p11-12;q32).
Thus, it may be possible that EBV can induce the
chromosomal translocations that are involved in defining
the specific subtypes of childhood ALL. However, to date
there is no substantive evidence to support such a hypothesis
but it may be an area that warrants further investigation.
A C677T polymorphism in the MTHFR gene was observed
to be protective with regard to infant leukemia with MLL
rearrangements, though not for childhood ALL
Q: What effect does this base change have on the activity
of the enzyme?
A: The base change from C to T in the MTHFR gene
causes an alanine to valine substitution at codon 222. This
amino acid substitution affects the catalytic MTHFR do-
main resulting in a thermolabile enzyme with reduced
catalytic activity. As stated in the manuscript, it is possible
that the affects on cancer risk of the polymorphisms in
MTHFR and related genes will be dependant on folate
status. Recently Krajinovic et al. (2003), also reported a
protective effect of the C677T and A1298C polymorphisms
in childhood ALL.
Although numerous earlier studies found a protective effect
of breast feeding on childhood ALL, the most recent data
are not confirmatory
Q: Could this reflect reduced commitment to unsupple-
mented and prolonged breast feeding, in recent years?
A: The latest reports are based on much larger numbers
and include in-depth reviews of earlier work. Differences
between populations in patterns of antigenic exposure of the
mother before and during pregnancy may also contribute to
differences between studies. Furthermore, the possible del-
eterious effects of breast feeding have not been as exten-
sively studied and it maybe that any protective effects are
outweighed.
114 T.J. Lightfoot, E. Roman / Toxicology and Applied Pharmacology 199 (2004) 104–117
Q: Or could it confirm the exposure-to-infectious-agents
hypothesis, since population isolation has become increas-
ing rare in recent decades?
A: For the reasons given above, we think that the
findings from earlier studies are probably less reliable and
should not be over-interpreted.
Q: Have any studies attempted to relate breast feeding to
risk of blood system cancers in offspring as adults?
A: Most work has been directed at examining associ-
ations between breast feeding and subsequent risk of
cancer in the mother. We could find no evidence to
suggest a link between breast feeding and risk of devel-
oping a haematological cancer as an adult. However,
Potischman and Troisi (1999) examined data relating to
in-utero and early life exposures in relation to risk of
breast cancer and found that a few studies have reported
that being breastfed as an infant may reduce the risk of
premenopausal breast cancer.
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