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Health
Scientific Committees
Scientific Committee on Veterinary Measures
relating to Public Health
Outcome of discussions
Report on
Public Health Aspects of the Use of Bovine Somatotrophin -
15-16 March 1999
INDEX
INTRODUCTION
EFFECTS OF rBST ON PUBLIC HEALTH
1. The rationale of risk assessment and risk
management in the context of public health
2. Public health aspect in terms of
safety of milk and milk products derived from rBST treated
lactating cows
2.1. Hazard identification
2.1.1. BST and its metabolites
2.1.2. IGFs
2.1.3. Additional hazards
2.2. Hazard characterisation: Qualitative and
quantitative evaluation of the nature of intrinsic biological
properties of IGFs
2.3. Exposure assessment: Occurrence and detection of
BST, rBST and IGF-I
2.3.1. Analytical methodology: State of the art in the
discrimination between non-treated and rBST-treated cows
2.3.1.1. GH and BST
2.3.1.2. IGF-I
2.3.2. Excretion of IGF-I in milk of non-treated and
treated (rBST) cows with particular reference to
physiological variation during lactation
2.4. Risk Characterisation: Bioactivity of GH and
IGF-I
2.4.1. Effects of rBST and IGF-I in the
Gastrointestinal Tract
2.4.1.1. Physiological properties and functions of
IGF-I in the gastrointestinal tract
2.4.1.2. Trophic effects of exogenous IGF-I:
2.4.1.3. Bio-availability of orally administered
IGF-I.
2.4.2. Systemic effects of rBST and IGF-I
2.4.2.1. rBST
2.4.2.2. IGF-I
3. Secondary risks related to the use of rBST in
animal production
3.1. Effect of rBST on drug metabolism in the target
animal species
3.2. rBST and clinical mastitis
3.3. Adverse effects related to alteration of milk
composition
4. Summary and Conclusions
5. References
5.1. Section A: Original Publications
_Toc446393172
5.2. Section B: Reports and opinion
statements
INTRODUCTION
Mandate
The Scientific Committee on Veterinary
Measures relating to Public Health is asked examine the use
of bovine somatotrophin (BST) to dairy cows as a
productivity aid to milk production. In particular the
Committee is invited to assess the possible direct and
indirect adverse effects on public health caused by the use
of BST under normal conditions.
In a parallel exercise, the Scientific
Committee on Animal Health and Animal welfare is asked to
report on the incidence of mastitis and other disorders in
dairy cows and on other aspects of the welfare of dairy
cows.
Background
Council Decision 94/936/EC of 20
December 1994 amending Decision 90/218/EEC concerning the
placing on the market and administration of bovine
somatotrophin (BST) prohibited the marketing and the use of
BST in the EU until 31 December 1999.
The Council asked the Commission to
entrust a Working Party of independent scientists with the
task of assessing the effects of using BST, in particular
as regards the impact of the use of this product on the
incidence of mastitis. In this request it is stated that
"BST is an issue which gives rise to considerable interest
among consumer, agricultural and industry interests. In
this context, concerns have been expressed about the safety
to humans, animals and the environment, the quality of
milk, the economic and social consequences in agriculture,
the climate for research and development, industrial
competitiveness and trade implications".
Comment
The present report is limited to the
public health aspects. The abbreviation BST is generally
used to indicate recombinant bovine somatotrophin
(rBST).
EFFECTS OF
rBST ON PUBLIC HEALTH
Products containing or consisting of
rBST are veterinary medicinal products within the meaning
of Directive 81/851/EEC on the approximation of the laws of
the Member States relating to veterinary medicinal
products. In the case of veterinary products derived from
biotechnology, Community concertation procedures
established by Directive 87/22/EEC have to be taken into
account as well, implying that the advice of the Committee
for Veterinary Medicinal Products (CVMP) must be obtained
before any decision on the authorisation of individual
products can be accepted. Recombinantly derived BST
products (rBSTs) may have slightly different chemical
structures from natural BST produced by the pituitary
gland, by adding a number of amino acids. Thus, each
product must be considered on its own merits by the CVMP
and it should be emphasised that it is not the aim of this
report to provide an expert opinion on certain veterinary
medicinal products.
In drawing up this report, the working
group has made use of previously compiled reports by
regulatory and advisory authorities in which aspects of the
safety, quality and efficacy of rBST products has been
examined. In particular, reference is made to:
1. 1st report concerning Bovine
Somatotrophin (BST) COM89, 379 final
2. 2nd report from the Commission tot
the Council and to the Parliament concerning Bovine
Somatotrophin (BST) SEC(91) 2521 final (16.01.1992)
3. CVMP-European Commission, DOCs. No.
III /3006-7/93, 23 January, 1993
4. FAO FNP 41/5: Food and Nutrition
paper: Residues of some veterinary drugs in animals and
foods. Bovine Somatotropins (1993)
5. Communication from the Commission to
the Council concerning Bovine Somatotrophin (BST) update
SEC(94), 1713 (25.10.1994)
as well as the recent
6. Report of the JOINT FAO/WHO Expert
Committee on Food Additives, presented at the 50th meeting
in Rome, 17/26.02.1998, (WHO: Food Additive Series 41, pp.
125-146, 1998)
7. Health Canada Report on BST
(1999)
8. Ongoing discussions in Codex
Alimentarius
In addition, recent scientific
literature, in particularly those which became available
after 1994 have been considered, as indicated in this
report (references section A). Finally a number of reports
and opinion statements have been considered as summarised
in section B of the references.
1. The
rationale of risk assessment and risk management in the
context of public health
Risk assessment and risk management are
not only scientific and technical activities, but also
represent a task attributed to science from the society. In
principle, risk assessment should represent a formally
defined and socially accepted evaluation process, which is
separate and independent from the decisions concerning risk
reduction or risk elimination. This separation and
independence was considered appropriate for preventing
possible biases in the risk assessment process, which could
be caused by influencing the desired neutrality of the
evaluation. Based on this principle, risk assessment should
be a matter of scientific evaluation, whilst risk
management should be a matter of political and social
decision making. Thus, additionally to the scientific
procedure of risk assessment the following issues may be
considered in risk management:
The perception of involuntary risk
factors (consumer's expectations and concerns).
The uneven distribution of risk and
benefits (e.g. health and/or economic advantage).
Risk assessment should cover the
following items:
1. Hazard identification
2. Hazard characterisation: dose
(concentration) - response (effect) assessment
3. Exposure assessment
4. Risk characterisation
As far as risk assessment is concerned,
the following definitions are applied:
Hazard identification
Identification of the adverse health
effects related to the intrinsic properties of a
substance.
Hazard characterisation
Qualitative and/or quantitative
evaluation of the nature of the adverse health effects.
This implies a dose (concentration) - response (effects)
assessment and an estimation of the relationship between
dose (or level of exposure) to a substance and the
incidence of a biological effect (response).
Exposure assessment
Qualitative and/or quantitative
estimation of the concentrations/doses to which human
populations (here: consumers) are exposed. Exposure
assessment requires information about the effects of
production, processing, handling, and consumption of
respective food commodities.
Risk characterisation
Estimation of the incidence and severity
of the adverse effects likely to occur to a human
population. Thus, the risk characterisation should "
include a qualitative and/or quantitative estimation,
including attendant uncertainties of the probability of
occurrence and severity of known or potential adverse
health effects" as specified in a document on "Risk
Assessment; Towards internationally acceptable standards
for food additives and contaminants bases on the use of
risk analysis" (Hugett et al., (1998).
In conclusion, risk assessment can be
regarded as scientific essentiality directed to provide
suitable answers to two questions:
1. What is the probability or likelihood
of an undesired event to occur, and
2. What are the consequences of this
undesired event in qualitative and quantitative
terms.
Thus, by definition, basic risk
assessment excludes in its initial phase concerns of the
decision making bodies and neglects the comparison and
balance of risk and benefits and societal requests related
to ethical, economic, technical and political aspects (EU,
1996, Technical Guidance Document in Support of the Council
Directive 93/67/EEC on Risk Assessment of New Notified
Substances and Council Regulation (EC) No 1488/94 on Risk
Assessment for Existing Substances).
2. Public
health aspect in terms of safety of milk and milk
products derived from rBST treated lactating cows
2.1. Hazard
identification
2.1.1. BST
and its metabolites
Growth hormone (GH, somatotrophin ST)
belongs to the protein family of somatolactogenic hormones.
In the 1980s advances in recombinant DNA techniques made
sufficient quantities of recombinant bovine growth hormone
(rBST) available for the use as milk production enhancing
agent. No therapeutic applications of rBST have emerged in
veterinary medicine (Burton et al., 1994).
The application of rBST to dairy cows
involves a parenteral application due to the instability of
BST in the gastrointestinal tract. Following application
and based on the peptide nature of rBST, rapid degradation
by cytosolic proteases and lysosomal enzymes which are
virtually present in all cells takes place. Residual
amounts of rBST may be expected at the site of injection
and in muscle and connective tissues especially following
improper administration of rBST formulations. The major
identified metabolite of rBST in plasma was the same as the
physiological thrombin cleavage product of BST. This was
demonstrated by sequence analyses in which two fragments
were found in a close to equimolar ratio. One sequence was
homologous to the N-terminus of the BST protein, whilst the
other sequence represented a fragment produced by cleavage
at the same site as the thrombin cleavage site of the BST
molecule (Bang et al., 1994b, Bang, 1995, Bang and Fielder,
1997). No formal risk assessment has been applied to these
cleavage products.
Hence there is no evidence that intact
BST or one of the above mentioned cleavage products exert
any direct biological effect after oral ingestion in humans
and in consideration of the heat-lability of rBST during
pasteurisation, non-specified ADI- and MRL values have been
considered for rBST (FAO/WHO Expert Committee on Food
Additives, Rome, 1998).
2.1.2.
IGFs
Elevated levels of pituitary growth
hormone are associated with increased liver secretion of
IGF-I and its binding proteins and chronic inhibitory
control of GH secretion is mediated by IGF-I feeding back
to all upper levels of the GH regulatory pathway.
Thus, in particular long-term metabolic
effects of GH or its analogues (rBST) are considered to
reflect the regulation of expression of certain genes. GH
regulated genes in the liver include the gene encoding for
IGF-I and recent work indicated that other tissues
including adipocytes and chondrocytes increase IGF-I mRNA
expression in response to GH. IGF-I has a high affinity for
a family of IGF-binding proteins, which modulate its
biological actions. Regulation by GH of these genes
encoding for binding proteins is considered as another
relationship between GH and IGF-I. In addition, several
other genes have found to be regulated by GH including the
spi2.1. gene, encoding a liver specific serine
protease inhibitor and the genes encoding cytochrome P450
enzymes (particularly the CYP2C family, see also section 3)
responsible for the biotransformation of numerous
pharmaceuticals and other xenobiotics (for review see
Carter-Su et al., 1996).
As the increase of circulating IGF-I
under the control of GH is considered as one of the
physiological mechanisms of GH, the application of rBST is
expected to induce the same mechanism. Indeed following the
zootechnical application of BST an increase in circulating
IGF-I concentrations has been found in lactating dairy cows
(for details see section 2.3.). Hence IGF's are single
chain polypeptides, they are excreted into milk. This has
been confirmed in different animal species including
humans. The amino acid sequence of IGFs is highly conserved
in mammals, and bovine and porcine IGF-I are identical to
human IGF-I (Honegger and Humbel, 1986, Francis et al.,
1989a,b), while IGF-II sequences exhibit a greater
variation among different animal species.
IGFs possess endocrine, paracrine and
autocrine activities. IGF-I acts as a progression factor in
the cell cycle and has mitogenic and anti-apoptotic
properties. IGFs are involved in numerous physiological
cell differentiation processes embodying for example
cellular differentiation in perinatal development as well
as processes such as maturation of ovary cells and regular
apoptosis, and cell proliferation. The numerous medical
reports (more than 1000 per year in the last two years)
focus on both aspects, the possibility of the use of IGF-I
in the treatment of distinct diseases, among others insulin
independent diabetes and renal failure, whilst others
describe the detrimental role of IGF-I as cellular growth
regulator and tumour promoter. The plethora of biological
effects exerted by IGF-I in vitro needs to be translated to
the complexity of mechanisms in the intact organism before
a final evaluation of dose-dependent effects can be
made.
2.1.3.
Additional hazards
In identifying the potential hazards,
secondary risks related to the use of rBST in dairy cows
need to be considered as well. These arise from possible
changes in milk composition of treated animals and
impairment of animal health, in particular the increased
incidence of mastitis resulting in a more frequent use of
antimicrobial substances (as discussed in more detail in
the report on the animal welfare aspects).
2.2. Hazard
characterisation: Qualitative and quantitative
evaluation of the nature of intrinsic biological
properties of IGFs
As it has been mentioned above, during
the last five years, an explosion of new information has
confirmed and extended the understanding of the pleiotropic
effects of the IGF system on growth, development, and
intermediary metabolism (Stewart and Rotwein, 1996). The
insulin-like growth factors (IGFs) comprise a conserved
pair of secreted proteins,
IGF-I (previously termed somatomedin C) and
IGF-II (termed somatomedin A). IGF-I is a
single-chain basic protein of 70 amino acids, and IGF-II is
a slightly acidic single-chain peptide of 67 residues
(Rinderknecht and Humbel, 1978a,b). By molecular cloning it
could be demonstrated that both IGFs are highly conserved
proteins found in an array of vertebrate species (for
recent reviews, see Rotwein, 1991, Dugay et al.,
1995).
Circulating IGFs are bound to carrier
proteins, denoted
IGF bindings proteins (IGFBPs). It soon became
evident that IGFBPs comprises a family of at last six
members, and a diversity of functions has been attributed
to these proteins, which prolong the half-life of
circulating IGFs, facilitate the transport of IGFs from the
circulation to the peripheral tissues, and thus potentiate
or inhibit IGF action (Bach et al., 1994; Jones and
Clemmons, 1995; Chan and Spencer, 1997; Hossner et al.,
1997; Lee and Giudice, 1997)
The cellular effects of IGFs are mediated
by two distinct receptors. The
IGF-I receptor (IGF-IR) is a hetero-tetrameric
glycoprotein which may be produced by mRNAs derived from a
single 21-exon IGF-IR gene, located on chromosome 15q25-q26
although several receptor variants have been described
(Abbott et al., 1992). The IGF-IR is similar in topography
and sequence to the insulin receptor and shares >50% amino
acid identity (Ullrich et al., 1986). The receptor is
composed of two ligand binding
a
-subunits and two transmembrane
b
-subunits. Ligand binding to the
a
-subunit triggers activation of the
intracellular tyrosine kinase, leading to receptor
autophosphorylation by an intra-molecular trans-mechanism
similar to that used by other receptor-tyrosine kinases
(Leroith et al., 1995).
Functional analysis of IGF-IR revealed a
complex signal transduction pathway as activation of the
IGF-IR by ligand binding causes not only rapid tyrosine
phosphorylation but also the intracytoplasmatic assembly of
a complex consisting of a variety of proteins
(SH2-containing proteins including Grb2, GAP, SH-PTP2, p85,
Nck and Sc), which link this receptor to the stimulation of
the protooncogene p21
ras and the mitogen-activated protein (MAP) kinase
pathway and thus overall regulation of gene expression
(Davis, 1994). Activation of phosphatidylinositol-3-kinase
via IGF-I signalling pathways leads to the induction of
several biological effects, including stimulation of
hormone-sensitive glucose transport (Cheatham and Kahn,
1995) and activation of the enzyme p70S6 kinase, which may
be involved in mitogenesis (Cheatham et al., 1995; Baserga,
1995).
Over-expression of human IGF-IRs in
mouse and rat fibroblasts has been found to induce
neoplastic transformation and development of tumours when
transfected cells were introduced into immunodeficient nude
mice (Kaleko et al., 1990). These findings indicate the
potential role for IGF-IR in tumour genesis (see
below).
Finally IGF-IR is involved in the
signalling pathway of other growth factors including
epidermal growth factor (EGF) and platelet-derived growth
factor (PDGF) (Coppola et al., 1994; Deangelis et al.,
1995) and at least two dominant oncogenes (large T antigen
of
simian virus 40 and the
ras and SRC oncogenes and tumour suppress genes
(Sell et a., 1993, Valentis et al., 1994, Sell et al.,
1994; Werner and Leroith 1995, Neuberg et al.,
1997).
The IGF-II receptor (IGF-IIR) is a single-chain
membrane-spanning glycoprotein that also is known as
cation-independent mannose-6-phosphate receptor. The IGF-IIR
is highly conserved among different species, with ~80%
identity being found among bovine, rat, mouse, and human
receptors (Kornfeld, 1992). The IGF-IIR is uniquely involved
in the clearance of lysosomal enzymes from the extra-cellular
environment. For example, the receptor plays a role in the
uptake of thyroglobulin after its secretion by thyroid
follicular cells and its subsequent degradation in lysosomes
(Herzog et al., 1987). It has been shown that IGF-IIR binds
the latent form of transforming growth factor-
b
1 (TGF-
b
1) and that this binding seems to be
essential for growth factor activation pointing to the role
of IGF-II in fetal development (Korner et al.., 1995, Lau et
al., 1994).
However, genetic studies have not
depicted a signalling function for the IGF-IIR and thus the
role for the receptor in mediating IGF-II actions remains
to be substantiated (Flaumenhaft et al, 1993, Korner et
al., 1995).
The physiological actions of IGF-I and IGF-II relate
to growth and development of the embryo and fetus and to
cellular differentiation, proliferation and cancer.
Over-expression of bovine, murine or rat
GH causes increased growth in transgenic mice accompanied
by two- to threefold elevations in serum IGF-I
concentrations (Mathews et al., 1988a). Transgenic mice
expressing human IGF-I in the liver and other tissues also
showed enhanced growth (Mathews et al., 1988b), while mice
over-expressing IGF-II did not (Wolf et al., 1994).
Over-expression and subsequent increase of serum IGF-I
levels manifest as selective organomegaly rather than
increase in skeletal size. This indicates that the effects
of GH or IGF-I on rate of growth on individual organs and
in the entire animal are not identical. IGF-I stimulates a
greater increase in kidney, spleen and thymus weight than
GH (Skottner et al., 1989). These qualitatively different
responses to GH and IGF-I might be related to the fact that
GH induces IGF-I synthesis in multiple tissues and also
enhances the expression of the major serum carrier protein
IGFBP-3 and its cofactor ALS (acid labile subunit) in
liver. The consequence of the induction of the expression
of this ternary complex is a more sustained exposure of all
tissues to IGF-I. IGF-I can stimulate the expression of
IGFBP-3 but has no effect on ALS synthesis.
Depending on the study, mice with a
disrupted IGF-I gene were significantly smaller in weight
and length than wild-type litter mates (Powell-Braxton et
al., 1993). Although this confirms that IGF-I and IGF-IR
are necessary for normal embryonic and fetal growth, IGF-II
seems to be essential. Despite numerous reports on IGF-II
gene expression and its regulation by parenteral imprinting
in rodents, comparable information from humans is scarce.
However, a concordant loss of imprinting of the human
IGF-IIR gene promoters has been found in certain cancers
(Zhan et al., 1995).
In conclusion, the results described
above, in conjunction with other known growth factor
signalling pathways and oncogene-mediated cell
transformation, provide the evidence for the role of IGFs
in tumorigenesis (Yang et al., 1993; Sell et al., 1995,
Minniti et al., 1995). However, when critically examining
this information it has to be concluded that IGF action is
involved in multiple biological processes thus rejecting
the possibility to define a dose-effect relationship which
describes all individual events.
2.3.
Exposure assessment: Occurrence and detection of BST,
rBST and IGF-I
Exposure assessment of food contaminants
comprises direct measurements indicating the presence and
quantity of the compound under investigation in certain
food commodities and molecular epidemiology providing
evidence of past exposure based on the analysis of typical
biomarkers (for example DNA- or protein adducts), or
selected somatic cell mutations, if appropriate.
Exposure assessment as applied to
chemically defined feed supplements or veterinary medicinal
products, e.g. compounds which are used on purpose
(intentionally) in food production processes comprises the
evaluation of the fate of the compound in the target animal
species (distribution and disposition of the parent
compound and its biological active metabolites) with the
aim to describe the time dependent (target animal) body
clearance and thus the quantity and likelihood of the
occurrence of residual amounts of the parent compound or
its biologically active metabolites in edible tissues, milk
and eggs.
rBST closely resembles the
physiologically expressed, endogenous bovine growth hormone
and is designed to exert the same effects as this natural
hormone in dairy cows. Thus, provided that rBST is used in
animal husbandry, two general questions need to be
addressed:
1. What is the state of art in
analytical methodology for the discrimination between
endogenous growth hormone profiles and zootechnically
applied rBST?
2. What is current knowledge on the
occurrence of residual amounts of rBST remaining at the
injections site and to what extend secondary, biologically
active metabolites such as IGFs are detectable in edible
tissues and milk as a consequence of rBST treatment.
2.3.1.
Analytical methodology: State of the art in the
discrimination between non-treated and rBST-treated
cows
2.3.1.1. GH
and BST
Formerly, the analytical methods used to
determine GH (bovine growth hormone; bovine somatotrophin
(bST)) concentrations in plasma, milk and tissue of cows
were exclusively radio-immunoassay procedures. None of them
were able to distinguish between the endogenous bST and the
recombinant growth hormone (rBST) products.
However, this assay was applied to
compare bST and IGF-I levels in tissues of control animals
and rBST treated animals (Choi et al., 1997). Although a
tendency towards a dose-related increase of tissue (muscle)
was observed, the differences between control animals and
rBST treated animals were statistically not
significant.
Since 1990 a number of interesting
developments have been launched. Electro-spray mass
spectrometry has been used to determine the differences in
molecular mass between the natural bST and one of the
recombinant products (Somagrebove®). Purified preparations
of bovine pituitary bST and rBST were used (Scippo et al.,
1997) and the accuracy of the technique was proven to be
about 0.05 % of the mass of the protein. This corresponds
to 11 Dalton for a protein of about 22000 Dalton, which is
more than enough to detect a difference of one amino acid,
as the average molecular mass of an amino acid is 115
Dalton. For rBST (Somagrebove®) a molecular weight of 22103
Dalton was measured, whereas the theoretical molecular mass
is 22094 Dalton. For the natural bST the mass spectrum is
much more complicated because theoretically four variants
exist. These have either 190 or 191 amino acids
(phenylalanine or alanine-phenylalanine at the N-terminal)
with a heterogenecity at position 127 (valine or leucine).
The two most dominant variants (190 and 191 amino acids
with leucine at position 127) give peaks corresponding to
molecular masses of 21725 Dalton and 21796 Dalton
respectively, whereas the theoretical values are 21720
Dalton and 21791 Dalton. For the detection of rBST treated
cows, the authors suggest to apply this technique on milk
and plasma samples after purification and concentration by
immuno-affinity chromatography. The minimum amount needed
to be obtained by this concentration steps is approximately
5 to 10 pmoles, which corresponds to 0.1 to 0.2 µg.
Considering that the minimal concentration of bST in plasma
is in the range of 1 ng/mL in non-treated cows, this means
that approximately a 100 mL plasma sample will be required
for analytical procedures as described.
Several attempts have been made to
measure bST concentrations in milk or in plasma by
non-radioisotopic immunoassays. A biotin-avidin sandwich
enzyme-linked immunosorbent assay for the determination of
bovine growth hormone in plasma has been developed by Secci
et al. (1988). Affinity-purified antibodies are immobilised
on microtiter plates. Bovine GH bound to the specific
antibody is then detected with a second anti-bovine GH
antibody labelled with biotin and peroxidase-conjugated
avidin. This method has a sensitivity as low as 0.25 ng/mL
plasma. No applications for the detection of rBST
administration are reported as of yet.
An avidin/biotin ELISA assay for bovine
somatotrophin is described by Zwickl et al. (1990). The
method uses affinity-purified polyclonal antisera raised in
rabbits to immobilize bST from blood or milk samples on the
wells of microtiter plates. Bound bST is quantitated by
adding biotinylated anti-bST antibody during the sample
incubation step, followed by incubations with horseradish
peroxidase labelled avidin D and ABTS substrate. Because
high-affinity anti-bST antibody is used, and the
biotinylated antibody is added directly to the sample, the
assay can be performed in less than 4 h while sensitivities
of 0.2 and 20 ng/mL in milk and blood, respectively, are
obtained.
Another competitive enzyme immunoassay
for bST was described by Hennies and Holtz (1993).
Antiserum, raised in rabbits, is preincubated with samples
and free antibodies from the reaction mixture are
immobilised using a microtiter plate coated with bST. Bound
antibodies remaining from the pre-incubation are visualised
using a biotinylated second antibody as a bridge for
subsequent amplification by an avidin-biotin-peroxidase
complex. The measuring range covers concentrations between
0.5 and 100 ng/mL. A similar competitive enzyme immunoassay
(EIA) for growth hormone in bovine pituitary cell culture
medium has been developed by Roth et al. (1997).
Ehrard et al. (1994) developed a
sandwich ELISA which is able to detect various rBSTs with
different N-terminal amino acids and thus allowed the
discrimination between rBST and pituitary bovine GH by an
affinity factor of 2.0. The authors believe that it might
be possible to identify rBST treated cows, but a field
study is needed for confirmation. No results of such a
field study have been reported as of yet. The other methods
for the control of treated and untreated animals are all
indirect methods. Various possibilities are under
development.
The injection of rBST into animals gives
rise to the production of antibodies against these
compounds. Their presence in plasma is an indirect proof of
the treatment, even after discontinuation of the treatment.
A first assay measuring these specific antibodies has been
elaborated (Scippo et al., 1997). ELISA plates are coated
with rBST and incubated with the cows serum. In the case of
antibodies present in the serum they are detected by the
addition of a second antibody against bovine IgG, coupled
to a peroxidase label.
2.3.1.2.
IGF-I
The second type of indirect methods are
related to the fact that GH and rBST application increase
IGF-I levels in milk. A radio-immunoassay for IGF-I in
bovine milk was developed by Zhao et al. (1991). The
technique was used for the analysis of milk samples
obtained from three control cows and three rBST-treated
cows (41-44 weeks post partum). Mean concentrations of
IGF-I were 2.77 ± 1.36 ng/mL in control cows and 3.30±1.40
ng/mL in treated cows, respectively.
The results of IGF-I quantitative assays
are controversial: the physiological reference values vary
from 1 to 30 ng/mL. This variation is not only based on the
reference populations of cows (inter-individual variation)
but also reflects the sensitivity of the antibodies applied
for the radioimmuno assays (Malven et al., 1987, Bang et
al., 1994b).
In conclusion, the analytical procedures
described, were designed to discriminate between treated
and non-treated animals. No formal intercomparisons of the
different analytical procedures are available allowing a
conclusive comparison of the reported IGF-I levels in milk
and dairy products.
An enzyme immuno-receptor assay for the
quantitation of IGF-I and insulin receptors in bovine
muscle tissue was developed by Boge et al. (1994). After
solubilization with Triton X-100 receptors were immobilised
in microtiter plates using receptor specific monoclonal
antibodies that recognise the intracellular beta-domain of
the respective receptors. The immobilised receptors were
labelled with either biotinylated IGF-I or insulin. The
bound ligands were detected with a streptavidin-horseradish
peroxidase technique. The assay, which was fully validated,
had a detection limit of 1 fmol receptor/well. The assay
system was used to study the effect of growth hormone
treatment upon IGF-I and insulin receptors in bovine
skeletal muscle. Three groups of 12 heifers (13 months old)
were treated with 320 or 640 mg rBST (slow release
preparation) every fortnight for 3 months. When samples of
the M.splenius were assayed for IGF-I and insulin
receptors, there was no difference between groups neither
with respect to receptor concentration nor affinity.
Finally, a third analytical procedure
was introduced, again designed to identify rBST treated
cows. This procedure is based on the fact that treatment
with rBST results in a decrease of the blood levels of
specific IGF binding proteins (IGFBP). The use of an
immunological method (Scippo et al., 1996) allows to
estimate this decrease. The concentration of IGFBP seems to
be 7 times lower in treated cows compared to untreated
animals. Thus, these methods would allow a reliable
identification of rBST treated animals.
2.3.2.
Excretion of IGF-I in milk of non-treated and treated
(rBST) cows with particular reference to physiological
variation during lactation
Evaluating more than 60 scientific
articles covering the period 1987-1998 it can be concluded
that mammalian milk contains various biological active
growth factors including IGF-I peptides (for review see Xu,
1998). In bovine milk, concentrations of IGF-I have been
observed in the range of:
1-34 ng/mL, normal milk (Malven et al., 1987;
Campbell & Baumrucker, 1989; Juskevich and Guyer, 1990;
Collier et al., 1991; Schams, 1991; Zumkeller,
1992).
100-300 ng/mL, colostrum (Francis and Read, 1986,
Malven et al., 1987; Campbell & Baumrucker, 1989;
Zumkeller, 1992).
4.3 ng/mL (range 1.3 - 8.1 ng/mL) average bulk tank milk
prior to BST use (Collier et al., 1991)
A comparison of retail milk originating
from 'labelled' milk (from non-treated cows) and
'non-labelled' milk (non-specified samples originating from
treated and non-treated cows) demonstrated a small,
insignificant increase of IGF-I concentrations in the
non-labelled milk samples (Eppard et al., 1994). However,
in this study the actual number of animals treated with
commercial rBST is not known.
During a lactation period, a typical
IGF-I profile in cow's milk varies from 150 ng/mL after
parturition to 25 ng/mL at the end of the first week of
lactation, to 1 to 5 ng/mL at day 200 of lactation
(Prosser, 1988; Xu, 1998).
In 1989, the first full report on milk
concentrations of IGF-I in cows treated with rBST appeared.
Prosser et al. (1989) showed a 3.6-fold increase in the
IGF-I concentration over a 7-day period of treatment. In
1994, Burton et al. highlighted several studies
demonstrating a two to fivefold increase of IGF-I as a
consequence of rBST treatment (Van den Berg, 1989;
Gluckman, 1990; Groenewegen et al., 1990; Juskevich and
Guyer, 1990).
A broad experiment comprising daily
injection and administration of a sustained release
formulation of rBST, respectively, was performed with 74
lactating cows (Zhao et al., 1994). Treatments began in the
fourth week of lactation and lasted 40 weeks. IGF-I was
monitored through early, mid- and late lactation. rBST
treatment resulted in a significant increase of plasma
IGF-I in all lactation periods for both treatment groups. A
higher milk IGF-I concentration, however, only occurred in
mid- and late lactation periods for the daily injection
group. It is worthwhile to mention that application of rBST
is restricted in most cases to the mid- and late
lactation.
The JECFA Report (1998) cites average
control values for IGF-I in milk of 3.7 ng/mL for untreated
cows, and a significant increase to an average of 5.9 ng/mL
as a consequence of rBST-treatment (see FAO FNP 41/5,
1993). Similarly, studies of different pharmaceutical
companies report an increase of IGF-I levels in milk
between 25 and 70 percent in individual animals (Burton et
al., 1994). Thus, the quantities present in the daily human
consumption of milk and dairy products are much lower than
the total amount of IGF-I secreted daily in the gut
(saliva, gastric juice, jejunal chyme, bile, and pancreatic
juice (Chaurasia et al., 1994, Bauman, 1995).
The IGF-I concentration in human breast
milk at weeks 6 to 8 is 22 ng/ml (Prosser, 1988). Likewise
to animals, IGF-I levels are high in the colostrum (17-30
ng/mL) and decline during lactation period (1-10 ng/mL)
(Xu, 1998 and references therein).
Milk secretions of mammals, however,
also contain amino acid N-terminally truncated forms of
IGF-I, which have a potency that is up to ten times greater
than normal IGF-I (Francis et al., 1988; Lemmey et al.,
1991). Regarding milk from cows, 3% of the IGF-I is
reported to be of the N-terminally truncated form
(Shimamoto et al. 1992).
Consequently, even at a 3% level, the
des(3N)IGF-I contributes substantially to an increase in
bioactivity.
Bovine IGF-I is not denatured by
pasteurisation (79
°
C for 45 seconds; Miller et al., 1989).
However, following processing of milk for infant formula
(121
°
C for 5 minutes) IGF-I is no longer
detectable (Collier et al., 1991). In contrast, an increase
of measurable IGF-I levels up to 70% following pasteurisation
have been reported as well (Juskevich and Guyer, 1990).
However, the different analytical methods applied allow no
direct comparison of these different reports. It is
worthwhile to mention here again that bovine IGF-I has been
shown to be identical in structure to human IGF-I (Honegger
and Humbel, 1986; Burton et al., 1994) as mentioned
before.
In conclusion, even though factors such
as stage of lactation, parity, level of nutrition and age
influence IGF-I levels in milk, the daily administration of
rBST will increase the concentration of IGF-I in milk
throughout the lactation period.
IGF-I in milk is resistant to
pasteurisation and even elevated levels of IGF-I have been
reported after pasteurisation. The latter might be related
to the standard analytical procedures which fail to detect
protein-bound (IGFBP-bound) IGF-I (see section 2.3.1.2.).
Consequently, consumption of milk from rBST treated dairy
cows will increase the daily intake of IGF-I.
2.4. Risk
Characterisation: Bioactivity of GH and IGF-I
2.4.1.
Effects of rBST and IGF-I in the Gastrointestinal
Tract
Although - at least theoretically -
measurable residual amounts of rBST may occur in edible
tissues (including the site of application) these residues
are not considered to be of public health concern as the
bovine growth hormone fails to interact with human growth
hormone receptors (
In contrast: human recombinant GH is under
investigation for therapeutic use in the treatment of
inflammatory bowel diseases).
Thus, even persistent rBST residues in
meat and milk are unlikely to be absorbed from the
gastrointestinal tract and would be biologically inactive
in humans. In addition, rBST in cow's milk is inactivated
by pasteurisation.
In contrast, IGFs are highly conserved
throughout mammalian species and bovine and human IGF-I are
identical. This implies that possible biological effects of
persistent and even slightly increased IGF-I levels in milk
(as discussed in section 2.2) have to be evaluated. The
following questions deserve attention:
- Does the IGF-I molecule remain
undestroyed in the gastrointestinal tract of humans (when
products from rBST-treated animals have been
consumed)?
- Based on the biological activity of
IGF-I activity as cellular growth factor and assuming that
IGF-I is not immediately destroyed in the gastrointestinal
tract, what is the consequence of the direct exposure of
the gut mucosa?
- What evidence can be provided that
orally ingested IGF-I enters systemic circulation and what
are the possible consequences of this systemic
bioavailability?
2.4.1.1.
Physiological properties and functions of IGF-I in the
gastrointestinal tract
Until 1991 little attention has been
focused on IGF actions in the gut (Read et al. 1991),
although it had been described earlier that particularly in
the fetal period the stomach contains one of the highest
concentrations of IGF-I mRNA and thus the IGF-I content of
the intestine exceeds that in liver. In human foetal
stomach and intestine IGF immuno-reactivity is localised in
epithelial cells with higher concentrations in the villus
than in the crypt cells. Adult rat intestines contain
slight to moderate IGF-I immuno-activity in scattered
epithelial cells covering the Peyer's patches. It was
concluded that gut expression of IGF-I and IGF-II is under
developmental regulation. IGF-II expression was found to be
maximal in foetal life declining rapidly in the early
postnatal period. This pattern parallels the postnatal
decline in liver IGF-II, but contrasts with the marked
increase in liver IGF-I in neonatal rats.
In addition, gut tissues express several
types of IGF bindings proteins including IGFBP-2 and
IGFBP-3. The expression pattern differs between stomach and
intestines.
Finally, IGF-I and IGF-II receptors have
been identified throughout the gut of several species,
including human, pig, rat and rabbit, again exhibiting
tissue-specific distribution patterns. Epithelial
receptor-binding activity is higher in the colon than in
other parts of the gastrointestinal tract, while receptor
density in the intestinal epithelium is greater in the
crypts than the villi. These findings suggest that receptor
expression declines with cellular differentiation (Read et
al., 1991 and references therein).
Evidence that exogenous supplementation
(via the intake of milk containing IGF-I) with IGF-I is
essential in the postnatal phase was provided by Dvorak et
al (1996). Applying a sensitive RT-PCR assay, IGF-I gene
expression was measured in different age groups (rats)
indicating 3 fold higher levels of IGF-I mRNA transcripts
in the rat small intestine of adults than in sucklings. The
authors concluded that the obvious limitation for IGF-I
synthesis in suckling rats may relate to significant
enteral IGF-I intake via milk. However, exogenous IGF-I
peptide as present in milk may be also responsible for the
down-regulation of IGF-I mRNA expression in the developing
rat gastrointestinal tract.
Of interest are also previous findings
in rats and pigs indicating high postnatal concentrations
of IGF-receptor specific mRNA in gastrointestinal tissues
relative to the mRNA concentrations of IGF-I (and IGF-II).
The temporal changes in IGF-receptor density have been
found to correlate with other indicators of intestinal
growth and functions (Schober et al. 1990, Burrin,
1997)
2.4.1.2.
Trophic effects of exogenous IGF-I:
In animals and humans there are specific
IGF-I receptors on the luminal surface of the
gastrointestinal epithelium (Donovan and Odle, 1994;
Zumkeller, 1992, Oguchi et al., 1997). IGF-I stimulates
growth and developments of the tissue and it has been
demonstrated that it increases cell proliferation in a
dose-dependent manner. Investigating the rate of cell
replacement in primary cultures of small intestinal
epithelium, Booth et al (1995) found a dose dependent
increase in epithelial growth at concentrations ranging
between 0 and 20 ng IGF-I per mL.
Initial experiments by Young et al.
(1990) had indicated that IGF-I administered either by oral
or parenteral routes, stimulated brush border enzymes
including maltase, lactase, alkaline phosphatase and
aminopeptidase, but had no effect on sucrase activity. In
contrast, IGF-II stimulated lactase and aminopeptidase, but
only by the oral route.
Comparative experiments in which the
effect of GH, IGF-I and GH plus IGF-I was measured
revealed, that all intestinal growth parameters were
increased following the administration of IGF-I and GH plus
IGF-I, whilst GH alone had no effect (Peterson et al.,
1997). These findings are in contrast to in vitro data in
which GH was found to significantly increase crypt
epithelial cell proliferation in explants of the human
small intestine (Challacombe and Wheeler, 1995; Wheeler and
Challacombe, 1997). However, this might be attributed to
indirect effects of GH as well, mediated by IGF-I.
Additional in vitro studies clearly
indicate the mitogenic nature of IGF-I on adult human
duodenal mucosa (Wheeler and Challacombe, 1997). The
trophic effects of IGF-I to increase crypt epithelial cell
proliferation in test explants, exceed those of GH and
insulin (Michell et al., 1997a). However, no comparative
studies have been conducted in vivo as of yet. As it could
be demonstrated that subcutaneous administration of IGF-I
improved mucosal structure and absorptive function after
small bowel transplantation in rats, the possibility to use
IGF-I therapeutically with the aim to improve adaptive
changes after surgical resections, has been discussed
(Sanderson 1997, Zhang et al., 1995; Chen and Nezu,
1997).
Taken together it can be concluded that
there is convincing evidence that IGF-I and other growth
factors excreted via milk play an important role in growth
and differentiation of gastrointestinal tract tissues and
support the concept of a physiological role of
colostrum-borne IGFs on the neonate (Baumrucker and Blum,
1993; Fholenhag et al., 1996; Fholenhag et al., 1997)). In
addition, clear evidence is provided that orally ingested
IGF-I reaches the receptor sites in the gut in its
biologically active form.
While the prominent role of IGF-I in the
modulation of somatic and gastrointestinal growth in the
neonatal was confirmed in several other experiments with
rats (Philipps et al., 1997, Steeb et al., 1997, Steeb et
al., 1995) and pigs (Burrin et al., 1996), it also became
evident, that oral administration of IGF-I results in
systemic effects (increase in body weight, liver and brain
weight) in suckling rats, and thus indicated the resistance
of IGF-I to degradation by gastrointestinal juices of the
suckling rat. Radio-labelled IGF-I, when administrated
orally remained receptor-active in gastrointestinal tract
tissue for at least 30 min post-ingestion (Philipps et al.,
1995).
The appearance of IGF-I in mammary
secretions has been shown to vary with physiological state.
Colostrum of all species contains high concentrations of
IGFs when compared with concentrations in mature milk
(Baumrucker et al., 1994). This implies that under
physiological conditions exposure to high levels of IGF-I
occurs only during the short perinatal period. The possible
trophic biological effects of a consistent IGF-I exposure
via milk throughout the entire life-span needs to be
established. Assuming a dose-dependent mitogenic effect of
IGF-I, the question remains to be answered, to what extend
exogenous IGF-I, being additive to the amount of IGF
physiologically present in the gastrointestinal tract (via
pancreatic and biliary excretions; Chaurasia et al., 1994),
is able to induce any adverse effect as a consequence of
long term exposure. This question needs to be addressed as
several in vitro studies indicated that IGF-I is mitogenic
to several colon carcinoma cell lines (Lahm, 1992; Michell
et al., 1997a; Guo et al., 1998)
2.4.1.3.
Bio-availability of orally administered IGF-I.
As IGF-I might be important in the
treatment of Laron dwarfism and insulin-resistant diabetes,
the oral application of recombinant (human) IGF-I has been
studied experimentally (the structures of human and bovine
IGF are identical). It could be demonstrated that the
initial low oral bioavailability of 9.3% could be increased
by the co-administration of aprotinin, and, more
importantly, by simultaneous application of casein. Casein
enhances the oral bio-availability of IGF-I in adult rats
to 46% and 67%, respectively (Kimura et al., 1997). The
orally administered IGF-I was present in the plasma as the
50-kDa and 150-kDa complexes, indicating that transmucosal
transport is facilitated by a specialised transport
mechanism.
These data confirm previous experiments,
in which an increase of the oral bioavailability of IGF-I
in the presence of milk casein had been reported in
neonatal calves and neonatal pigs (Xu and Wang, 1996;
Vacher et al., 1995), whereas other studies report a poor
absorption rate only (Donovan and Chao, 1997). These
experimental data allow the hypothesis that IGF-I possess a
considerable oral bioavailability also in humans after
consumption of IGF-I enriched milk as the casein acts as
inhibitor of several proteases (Playford et al., 1993).
This hypothesis needs to be reflected in the light of
epidemiological studies indicating a positive correlation
between dairy product consumption and breast cancer risk
(see section 2.4.2.2.; Del Guidice et al., 1998).
2.4.2.
Systemic effects of rBST and IGF-I
2.4.2.1.
rBST
Although recombinant BST has been
considered "essentially chemically the same as natural
bovine growth hormone", certain specific differences are
worthwhile to mention:
Recombinant rBSTs differ from the
natural growth hormone by 1-9 amino acids. In most cases,
the N-terminal alanine is replaced by methionine. Dairy
industry experiments indicated that the additional,
terminal methionyl residue makes rBSTs more immunogenic
(FDA Veterinary Note, 1988).
Short-term studies provided no evidence
of carcinogenic properties of rBST in Rhesus monkeys.
Although the study design is questionably these data fit
into the general concept of species-specificity of peptide
hormones. However, the possibility that growth hormone
cleavage products might retain certain biological
properties including the stimulation of the production of
growth factors like IGF-I has never been properly
addressed.
Furthermore, the role of other milk
constituents, which might be altered in their relative
concentration in milk, requires further evaluation as not
only milk fat quantity and composition is modified by rBST
administration but also an increase in the excreted amount
of IGF-I, truncated IGF-I ((des3N-IGF-I) and IGFBPs in
bovine milk has been reported (Shimamoto et al, 1992;
Groenewegen et al., 1990) (see also section 2.3.2.).
2.4.2.2.
IGF-I
Previous epidemiological studies have
indicated a positive correlation between dairy product
consumption and breast cancer (for review see Outwater et
al., 1997).
Detailed analyses on the relative risk
(RR) including adjustment of RR coefficients for age at
first birth and economic variables provided further
evidence that milk and cheese were the only dietary
variables to remain significantly positive. It was
concluded that the relative risk of breast cancer increases
with the amount of dairy products consumed; this trend was
not evident with respect to meat consumption.
Hence
in vitro studies indicated that IGF-I is a potent
mitogen for breast cancer cells, the link between milk
IGF-I concentrations and the relative risk for human breast
cancer was established. This hypothesis is supported by the
fact that human and bovine IGF-I are identical (as
mentioned before) and also IGF-I in milk is present in its
unbound form.
These mitogenic effects on cell
proliferation rate of breast cancer cells could be observed
at concentration as low as 1 ng/mL (Zapf et al., 1981). The
average concentration in milk varies between 1-34 ng/mL
(see section 2.3.2.).
Nearly all breast cancer cell lines and
breast cancer cells from fresh tumour biopsies have
receptors for IGF-I, and IGF-I binding to both, benign and
metastatic human breast tumours is increased compared to
normal mammary tissue binding (Macaulay, 1992; Peyrat et
al., 1992; Jammes et al., 1992). In addition, highly
malignant human breast cancers produce and secrete IGF-I.
This observation has been used as diagnostic tool in
clinical oncology but also indicates that IGF-I might be
directly involved in tumorigenesis. IGF-I causes changes in
the cell cycle and activates oncogenes such as
c-fos (Li et al., 1997). Evidence suggests also that
oncogenes may encode IGF-IRs whose over-expression seems to
be involved in the transformation from natural mammary
tissue growth to breast cancer (Kaleko et al.,
1990).
As IGF-I receptors are over-expressed in
virtually all breast cancer cell lines they are considered
to be related to enhanced proliferation whilst inhibiting
programmed cell death (apoptosis). Recently, Resnik et al.
(1998) could demonstrate that IGF-IR expression was 14-fold
higher in malignant breast tissue than in normal breast
tissue and receptor function, as demonstrated by kinase
activity, was 2-4 fold higher in purified receptor
preparations from malignant breast tissue.
Epidemiological data stressing the role
of IGF-I in breast cancer became available with the nested
case-control study within the prospective Nurses' Health
Study (Hankinson et al., 1998). This well-known study
started in 1976 and includes women of different ages
(including pre-menopausal and post-menopausal cohorts).
Plasma concentrations of IGF-I and IGFBP-3 were measured in
blood samples collected in 1989-1990. These IGF-I
concentrations were compared by logistic regression with
adjustment for other breast cancer risk factors.
A positive relation between circulating
IGF-I concentration and risk of breast cancer was found
among pre-menopausal women (top
vs bottom tertile: relative risk 2.33 (1.06 - 5.16)
with
p for trend 0.08; selecting pre-menopausal women
less than 50 years old, the relative risk amounted to 4.58
(1.75 - 12.0) with
p for trend 0.02). After adjustment for plasma
IGFBP-3 concentrations, the relative risks increased to
2.88 and 7.28, respectively. Neither in post-menopausal
women nor among the whole study group, a comparable
association between circulating IGF-I concentration and
breast cancer could be established (Bohlke et al.,
1998).
Del Giudice et al., (1998) found in
another case control study a positive association between
IGFBP-3, circulating insulin levels and the incidence of
pre-menopausal breast cancer. These recent studies confirm
previous case control studies, also reporting a positive
relation between plasma IGF-I concentration and breast
cancer risk (Bruning and Clemmons., 1995; Peyrat et al.,
1993). However, it should be taken into account that the
recent studies are also in favour of the suggestion that
plasma IGF-I concentrations are an early marker in the
identification of women at high risk, rather than
indicating a causal relationship between cancer incidence
and circulating IGF-I levels. In addition, these
epidemiological data suggest a correlation between IGF-I
and IGFBP-3, however, the individual contribution to the
overall bio-activity in the tissues remains unclear.
Further evidence for the relation
between IGF-I and breast cancer originates from experiments
with rodent species. Energy restriction can decrease tumour
development in multiple models. As energy restriction also
lowers IGF-I levels, thereby favouring apoptosis over cell
proliferation, energy restriction slows tumour progression.
Recent studies (Dunn et al., 1997) confirmed this
hypothesis as the protective effect of energy restriction
could be abolished by supplementation of IGF-I.
The responsiveness of breast epithelial
cells to IGFs is modulated by estrogens and estrogens
appear to act at several points of the IGF signal
transduction and to regulate both, IGF-I and IGF-II
expression as well as IGF binding proteins and type I IGF
receptors (Westley et al., 1998; Koval et al., 1998). These
data confirm previous studies describing that estrogens
increase the level of IGF-I in human breast tissue (Osborne
and Arteaga, 1990). Furthermore, IGF-I stimulates estrone
sulphatase activity (Purohit et al., 1992) and the number
of IGF-I receptors has been found to be positively
correlated with the number of estradiol receptors,
suggesting synergistic mechanisms (Peyrat et al.,
1992).
It is worthwhile to mention that in
breast cancer as well as in prostate cancer, bladder
tumours, gastric cancer and paraganglioma tumours an
increased expression of IGF-II was demonstrated providing
further evidence for the role of IGFs in autocrine cancer
cell growth
in vivo (Li et al., 1998).
Finally, IGF-I has been found to be a
mitogen for prostate epithelial cells. A prospective case
control study of men, participating in the Physician's
Health Study revealed a strong positive association between
IGF-I levels and prostate cancer risk (Chan et al., 1998;
Brower, 1998). Relative risk (RR) varied in an univariate
analysis between 0.62 and 4.74 with
p for trend of 0.006 (test for linear trend
calculated by assigning the medians of the quartiles as
scores). Multivariate analysis (with simultaneous
adjustment for IGF-I or IGFBP-3) revealed quartiles
associated RR values between 0.83 and 10.6 with a
p for trend of 0.001.
Again the question remains to be
answered whether or not an increased level of circulating
IGF-I has to be considered an early marker, predicting
prostate cancer risks, rather than indicating a causal
association.
3. Secondary
risks related to the use of rBST in animal
production
Based on the nature and intrinsic
activity of GH in the target animal, a number of secondary
effects can be anticipated:
3.1. Effect
of rBST on drug metabolism in the target animal
species
Growth hormone has been shown to exert
its biological effect by regulating the expression of
different genes, including the expression of enzymes of the
cytochrome P450 family.
Particular reference is made to the
CYP2C family, which comprises a considerable percentage of
total P450 activities in bovines. CYP2C is involved in the
bio-transformation of a wide range of pharmaceuticals
facilitating their bio-inactivation and elimination.
Down-regulation of CYP2C would result in delayed body
clearance and increase the biological half-life of these
drugs (Witkamp et al., 1993, Chilliard et al., 1998). This
comprises a virtual risk towards an increase of undesirable
residues in edible tissues and milk and might lead to an
intensified drug residue monitoring.
3.2. rBST
and clinical mastitis
The use of rBST might comprise the risk
of an increased incidence of mastitis in dairy cows (for a
detailed discussion of this item we refer to the
corresponding report devoted to Animal Welfare aspects).
The public health and food safety aspects of mastitis in
dairy cows are exclusively associated with the potential
problems of side effects from using antimicrobials in the
treatment or prevention of such cases. Treatment of
clinical mastitis cases with antimicrobials is not limited
to those cases which may be classified as
severe, although such cases are probably more likely
to receive systemic treatment. Also
mild clinical cases are often treated with local
application of antimicrobials, such as the application of
formulations for intra-mammary use. Even cases of
sub-clinical mastitis are sometimes treated with
antimicrobials, depending on other factors in the herd, as
are cows being dried off before calving (Radostits et al.,
1994). The result is that mastitis is the one condition in
dairy cows which is associated with use of the largest
amount of antimicrobials. It is therefore not surprising,
that by far the most frequent reason for residue violations
in milk are related to mastitis treatment (Leslie and Keefe
1998). This applies in particular in cases where the
principles of Good Clinical Practice are not
respected.
The public health reasons for limiting
as far as possible the use of antimicrobials in dairy cows
are the risk of:
- an increased incidence of allergic
reactions from drugs and their metabolites in consumers of
milk and dairy products;
- an increased selection of bacteria
resistant to antimicrobials.
Allergic reactions:
It is estimated that 3 - 10% of the
human population is allergic to penicillin and other
beta-lactam antibiotics, which constitute the most common
therapeutic treatment for clinical mastitis. There are a
few reported cases in the literature on allergic reactions
following consumption of contaminated milk.
There is no available data on how the
risk of such residues vary with occurrence of mastitis in
the source cows, but as a general assumption one may
consider that increasing risk of mastitis which is treated
by antimicrobials will increase the risk of such residues
(Kaneene and Ahl, 1987).
The extent to which this risk is
modified or prevented by testing for residues by routine
monitoring is also not known, but of course any violation
which is detected before the milk is processed will lower
the risk of residues in milk for consumption. The test
characteristics (sensitivity and specificity) of the test
procedures used for detection of residues will, therefore,
influence the outcome of the monitoring, and critical
evaluations of some of the tests used have been published
(Gardner et al. 1996).
Antimicrobial resistance:
The risk of antimicrobial resistance
following veterinary, including mastitis related, use of
antimicrobials is the subject of another scientific report
currently being prepared by the Scientific Steering
Committee. Recent publications referring to the specific
issue of bacterial resistance following mastitis related
use of antimicrobials vary in their evaluation of the
phenomenon (Hillerton 1998, Sandgren 1998, Wegener 1998,
Aarestrup and Jensen 1999). The issue of antimicrobial
resistance in general is subject of several ongoing
evaluations in the EU and Codex Alimentarius.
It can be anticipated that with an
increase of the incidence of bovine mastitis more
veterinary medicinal products will be used. This practice
comprises a virtual risk toward an increase of undesirable
residues in milk and other edible tissues and might lead to
an intensified drug residue monitoring program within the
European Community. Furthermore, the increased use of
antimicrobial substances in the treatment of rBST related
mastitis might lead to the selection of resistant
bacteria.
3.3. Adverse
effects related to alteration of milk composition
Several reports express concerns about
undesirable allergic reactions which might occur after the
consumption of milk obtained from rBST-treated cows.
Previously, the antibody response to rBST has been
investigated as indirect measure of the possible absorption
of rBST from the (rat) gastrointestinal tract. However, the
question whether or not a change in milk protein
composition as a consequence of rBST application to dairy
cows might pose an additional risk factor in the
development of food allergies has so far not been addressed
adequately.
4. Summary
and Conclusions
Numerous reports have indicated that the
application of recombinant growth hormones (rBST, rbST)
increases productivity of dairy cows measured as total milk
yield per animal per lactation period. The application of
rBST therefore may result in economic benefits although no
therapeutic indications have been considered in the target
animal species to date.
Based on its peptide nature, rBST has to
be applied parenterally and the concept of species -
specificity implies that residual amounts of unchanged rBST
fail to induce a biological response in species (including
humans) other than bovines. However, the nature of rBST
cleavage products and their biological activity has not
been investigated in detail.
Comparably to the endogenous growth
hormone, rBST is known to increase the level of circulating
IGF-I in the target animal followed by an increased
excretion of IGF-I in milk. Consequently increased levels
of IGF-I in milk have to be included in the estimation of
potential health hazards originating from the zootechnical
use of rBST.
IGF-I is a physiological constituent of
bovine milk. Data on the actual amount of IGF-I in milk are
inconsistent as physiological levels show a considerable
variation depending on the age of the animals, state of
lactation and nutritional status. The highest
IGF-concentrations in milk are found at the initial phase
of lactation (colostrum) and decline as lactation
progresses.
The various analytical techniques for
the determination of IGF-I and its truncated forms need to
be evaluated in validated procedures. Present data do not
provide a conclusive answer to whether or not previously
applied analytical techniques have underestimated the
actual IGF-I level in milk by neglecting the protein-bound
fraction, and to what extent the ratio between free and
bound IGF-I in milk has changed as a consequence of rBST
treatment resulting in a relative increase of the free
IGF-I fraction.
Application of rBST increases the amount
of excreted IGF-I in milk by 25-70 % in individual animals.
The Committee noted that bovine milk may contain truncated
IGF-I (des(1-3)IGF-I) which was found to be even more
potent than IGF-I in the anabolic response when given
subcutaneously to rats. No quantitative data are available
indicating the additional level of this truncated form of
IGF-I in milk from rBST-injected dairy cows.
The biological activity of IGF-I
comprises endocrine, paracrine and autocrine effects and
IGF-I has been identified as cellular growth factor with
mitogenic, anti-apoptotic properties and may thus directly
interfere with physiological mechanisms involved in the
removal of transformed cells. Evidence on the physiological
essentiality of IGF-I in foetal and perinatal development
is accumulating. Biomedical research focuses on the
possible use of IGF-I in the therapy of distinct diseases,
whereas the detrimental role of IGF-I in tumour progression
is disputed.
Experimental evidence for an association
between IGF-I and breast and prostate cancer is supported
by epidemiological studies. The bimodal activity of IGF-I
being essential in the process of cellular differentiation
regulating the expression of several genes, and acting as
cellular growth factor with anti-apoptotic properties
hinders the definition and establishment of a
no-adverse-effect level, a paradigm in conventional risk
assessment.
Advocates of the medical (therapeutic)
use of IGF-I refer to the short half-life and the
auto-regulatory mechanisms sequestering free biologically
active IGF-I via endogenous binding proteins
(IGFBPs).
Opponents refer to the epidemiological
evidence arising from the recently published cohort studies
indicating an association between circulating IGF-I levels
and the relative risk of breast and prostate cancer,
respectively.
Elevated plasma IGF-levels may be
considered as a predictive marker for breast and prostate
cancer. However, it should be emphasised that all these
epidemiological studies refer to a time interval in which
exposure to dairy products originated exclusively from
non-rBST treated animals. Whether or not the use of rBST
will modify the level of risk, remains to be
substantiated.
Following the globally accepted concept
of risk assessment it is concluded that:
- Direct risks associated with the use
of rBST in dairy cows appear to be related to the possible
increase of IGF-I levels in milk. The diverse biological
effects attributable to the intrinsic activity of IGF-I,
exerting a broad variety of metabolic responses through
endocrine, paracrine and autocrine mechanisms, make the
definition of an in vivo quantitative dose-effect
relationship virtually impossible.
- Risk characterisation has pointed to
an association between circulating IGF-I levels and an
increased relative risk of breast and prostate cancer. In
addition, the possible contribution of life span exposure
towards dietary IGF-I and related proteins, present in milk
from rBST treated cows, to gut pathophysiology particularly
of infants, and to gut associated cancers need to be
evaluated.
- The available data basis for exposure
assessment, i.e. the amount of IGF-I and/or its truncated
forms excreted in milk following the administration of rBST
to dairy cows, is incomplete.
In addition secondary risks associated
with the use of rBST in dairy cows are:
- Potential changes in milk protein
composition which might favour allergic reactions.
- An increased use of antimicrobial
substances in the treatment of rBST related mastitis which
might lead to an increased risk of residue formation in
milk and to the selection of resistant bacteria.
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6-10
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