September 1997
Potential Public Health Impacts Of The Use Of Recombinant Bovine Somatotropin In Dairy Production
by Michael Hansen, Ph.D., Jean M. Halloran, Edward Groth III, Ph.D., Lisa Y. Lefferts
Prepared for a Scientific Review by the Joint Expert Committee on Food Additives
Introduction: Goals of the JECFA Review
The task of assessing the safety of widespread commercial use of recombinant bovine somatotropin (rbST) is far more complex and difficult than assessments of most food additive safety questions. In this case there is no food additive involved, nor is the central issue residues of rbST itself in meat or milk. Establishment of a Maximum Residue Level (MRL) for rbST is not appropriate.
The central human health questions are, to what extent does rbST use increase the level of the hormone Insulin-like Growth Factor I (IGF-I) in milk, and what possible risks to public health might be associated with consuming milk with increased IGF-I levels? We present below a review of what we believe is sufficient credible evidence that average IGF-I levels are increased in milk from rbST-treated cows. We present further recent evidence that IGF-I survives digestion and passes into the intestinal tract, and we review evidence that associates exposure of the intestinal epithelium to IGF-I at levels within the range found in milk with cellular growth responses linked to the risk of colon cancer. While this evidence is provocative, it falls short of providing an adequate basis for a quantitative risk assessment.
Even if JECFA were to agree that there is sufficient evidence to presume that increased IGF-I levels in milk do pose some risk to public health, setting an MRL for IGF-I is neither appropriate nor feasible. IGF-I occurs naturally in milk and natural levels vary. An MRL set high enough to allow for natural fluctuations in IGF-I levels in milk from cows not treated with rbST would inevitably also allow most increased levels resulting from rbST use. An MRL set high enough to accommodate the upper limit of natural variation would allow average IGF-I levels to increase substantially, resulting in significantly increased consumer exposure to IGF-I. It does not appear feasible, therefore, to use an MRL approach to limit increased total public exposure due to a gradual rise in average IGF-I levels in milk.
If the outcome of JECFA’s review of this issue is not to establish an MRL, what should it be? There is a critical need for a scientifically rigorous and objective, balanced and non-partisan assessment of potential public health risks of rbST use. Such an assessment needs to summarize lucidly for the many policymakers facing this issue both what is known scientifically and, equally important, what is as yet not known, but needs to be known to better assess possible risks. Rather than make a "forced choice" between declaring rbST use "unsafe" because there is sufficient credible evidence of risks, or "safe" because the evidence of risks is judged less than sufficient, JECFA should aim to produce a definitive answer to the following questions:
- What changes in the composition of milk are very likely or somewhat likely to occur as rbST use becomes widespread? (For example, increases in average IGF-I levels? Increased likelihood of antibiotic residues?)
- What are the nature (type of possible adverse effects) and magnitude (number of people exposed to risk, existence of sensitive subpopulations, quantitative estimates of risk) of possible public health effects associated with those likely changes in milk composition?
- What other public health and economic risks, such as increased antibiotic resistance, or increased susceptibility of rbST-treated cows to bovine spongiform encephalopathy, may be associated with rbST use?
- For each specific issue addressed, which questions can be answered definitely, based on sufficient data of good quality? Which can be answered tentatively, based on some good data, but require additional research to resolve definitely? Which questions are scientifically reasonable to ask, based upon limited existing data or theory, but cannot be answered with available evidence?
In this paper, we summarize new evidence, and interpret it in the context of the evidence as a whole, on three key issues. They are: 1) Do IGF-I levels in milk from rbST-treated cows pose a potential human health hazard? 2) Does use of rbST increase mastitis incidence and thereby lead to increased antibiotic residues in dairy products and/or exacerbate problems of antibiotic resistance in bacteria? and 3) Does rbST use potentially exacerbate BSE risks?
1. Do IGF-I levels in milk from rbST-treated cows pose a potential human health hazard?
IGF-I levels in milk are important because IGF-I is the hormone that actually mediates much of the cellular response to growth hormones in cows and humans. JECFA previously concluded, and data published in 1986 demonstrated that, while human and bovine growth hormones differ by up to 35% in their amino acid sequences, human and cow IGF-I are identical (Honegger and Humbel, 1986). An assessment of potential public health impacts of IGF-I in milk requires a review of the evidence on three subsidiary questions: Do IGF-I concentrations increase in milk from rbST-treated cows? Does IGF-I survive digestion? And, What are the possible adverse health effects of increased IGF-I in milk?
Do IGF-I concentrations increase in milk from rbST-treated cows?
JECFA addressed this question in its 1993 report and concluded that the best studies then available did not indicate an increase in average IGF-I levels. However, a study by Monsanto did not become available to the public until after JECFA completed its review. We have conducted a new analysis of the data, considering both the latest Monsanto study and all earlier evidence on this question. We conclude that the weight of evidence indicates that rbST use does increase IGF-I levels in milk, substantially. In our judgment, the most important question is no longer whether IGF-I levels increase, but rather how much they increase, on average.
The latest Monsanto study, and five of the seven studies previously reviewed by JECFA, found an increase in IGF-I levels. In five of these six studies, the increase was statistically significant, and the sixth study, involving a very small number of test subjects (six rbST-treated cows and six controls), found an increase that was not statistically significant. The three studies that did not find a statistically significant increase used the lowest doses of rbST. All four Monsanto studies, using the dose recommended by Monsanto to farmers, did show a statistically significant increase. We discuss all eight studies in more detail below.
The newly available Monsanto study was conducted at the Monsanto Animal Research Center in O’Fallon, Missouri and reported in late 1993 in the US FDA’s Freedom of Information Act Summary of the data used to gain approval for POSILAC, Monsanto’s rbST product. This study involved 18 cows, an rbST dosage of 500 mg injected every 14 days, with milk collected 7 days after each of three injections. IGF-I levels were statistically significantly elevated in milk from rbST-treated cows. Indeed, the milk IGF-I levels of treated and control cows did not even overlap, i.e. the milk IGF-I level from the 9 rbST-treated cows was higher than any of the levels found in the milk of control cows: "During the study, milk IGF-I concentrations ranged from 3.16 to 3.35 ng/ml for control cows and from 3.49 to 5.31 ng/ml for treated cows. The difference in milk IGF-I between control and treated cows was statistically significant at the 5% probability level" (FDA, 1993: 121).
The first Elanco study (Schams and Karg, 1988a,b) involved 8 cows and rbST dosages of 640 mg injected every 28 days, with milk collected at 2-3 day intervals after the third and fourth injections. The study found that "After somidobove injection, mean IGF-I levels in the treated animals are always higher than those found in controls. The average IGF-I milk concentration found in control animals was 28.4 ng/ml, and the average IGF-I milk concentration in the 640 mg somidobove-treated animals was 35.5 ng/ml. Therefore, in this study an increase of approximately 25% of the mean was found in the somidobove-treated animals" (FAO, 1993: 120-121). The 25% increase was statistically significant (Juskevich and Guyer, 1990).
A second Elanco study (Davis et al., 1989) involved 36 cows and rbST dosages of either 320 mg or 640 mg injected every 28 days, with milk collected 4 times (days 3, 10, 17 and 24) after the first injection. They found that "the concentration of IGF-I in milk was higher by day 3 in cows treated with 320 and 640 mg of somidobove relative to the control cows. The values at day 10 and thereafter were not statistically different between treatment groups. . . . After somidobove treatment in this study, the levels of IGF-I in the milk increased less than 50% relative to the milk IGF-I content in the control cows" (FAO, 1993: 121). Thus, long-term average IGF-I levels increased, but were not statistically significant.
A third Elanco study (Coleman et al., 1990) involved 12 cows, an rbST dosage of 640 mg injected every 28 days, with milk collected 4 times (days 3, 10, 17 and 24) after the first and second injections. There was not a statistically significant increase in milk IGF-I concentrations in treated animals.
The sole American Cyanamid study (Schingoethe and Cleale, 1989) involved 20 cows and an rbST dosage of 10.3 mg injected daily, with milk collected weekly for 16 weeks. This was a very small doseless than a third the dose used in the Monsanto studies. Further complicating the test was the fact that it also looked at the effects of diet composition. Animals were fed a normal diet or a high energy and protein diet and got injections of either rbST or a placebo, so there were four treatment groups. The study found that "mean concentration throughout the study in control animals was 9.67 ng/ml and in the somagrebove-treated animals was 9.06 ng/ml. Concentrations of IGF-I were significantly higher (P < 0.05) in the milk from the cows that consumed the high energy and protein diet than those that received the normal diet. Therefore this study demonstrates that there is no increase in IGF-I concentration in the milk of cows treated with up to 10.3 mg of somagrebove when tested for a 16 week period" (FAO, 1993: 123).
The first Monsanto study (Torkelson et al., 1988) involved 18 cows and an rbST dosage of 500 mg injected every 14 days, with milk collected 7 days after each of 3 injections. The study found that "After each of the 3 doses, mean milk IGF-I in controls was 3.22, 2.62 and 3.78 ng/ml and in treated cows was 3.80, 5.39 and 4.98 ng/ml, respectively. Differences between treated and control groups was [sic] significant after the second and third doses" (FAO, 1993: 121). Thus, the average IGF-I concentrations were increased by 18%, 106% and 31.7% for injection cycles 1, 2, and 3, respectively in the treated groups compared to controls.
The second Monsanto study (White et al., 1989) involved 18 cows and an rbST dosage of 500 mg injected every 14 days, with milk collected 7 days after each of 3 injections. As in the Torkelson et al. study, mean milk IGF-I concentrations were statistically significantly higher after the second and third doses. Mean milk IGF-I in controls was 3.17, 3.34 and 3.35 ng/ml and in treated cows was 3.50, 5.33 and 4.68 ng/ml, respectively. Thus, the average IGF-I concentrations were increased by 10%, 60% and 40% for the first, second and third doses, respectively in the treated group compared to controls.
The third Monsanto study (Miller et al., 1989) involved 64 cows and an rbST dosage of 500 mg injected every 14 days, with milk collected 7 days after each of 10 injections. This was both the longest of the eight studies (140 days) and the largest (64 cows). We therefore regard it as the most definitive and comprehensive of the eight studies. The study found that "milk concentration of IGF-I was increased across the 10 injection cycles" (FAO, 1993: 126). For primiparous cows the increase was 74%, from 3.5 ng/ml to 6.1 mg/ml for control and rbST-treated cows, respectively. For multiparous cows, the increase was 41%, from 3.9 ng/ml to 5.6 mg/ml for control and rbST-treated cows, respectively. Both results are statistically significant.
Table 1 summarizes the data from all eight studies. Six of the eight studies found that IGF-I levels were higher in milk from rbST-treated cows compared to control cows, with five showing statistically significant increases ranging from 25% to 74%. The three studies that found no significant increase in IGF-I in milk from rbST-treated cows involved the lowest dosages of rbST. This becomes clear if the dosages are all expressed as mg rbST/14 days. The American Cyanamid study used only 10.3 mg/ day, equivalent to 144 mg/14 days (Schingoethe and Cleale, 1989), while the Elanco studies used either 320mg or 640 mg/28 days, equivalent to 160mg or 320 mg/14 days. The one Elanco study that did find a statistically significant increase had the second longest experimental period (118 days), which may explain why the results were statistically significant (the effect might only start to appear after some time period).
The four Monsanto-sponsored studies all found statistically significant increases and used the highest dosage of rbST (500 mg/14 days), which was from 50 to 350 percent higher than the dosages used in the other studies. Furthermore, of the eight studies, the Miller et al. 1989 study had the largest sample size (64 cows) and the longest duration of experiment (10 injection cycles, or 140 days) (Table 1). On the basis of sample size, duration of experiment and rbST dosage used, Miller et al.’s 1989 study is clearly the "most definitive and comprehensive" of the studies. It found that rbST treatment led to a statistically significant increase in milk IGF-I levels (74% and 41% for primiparous and multiparous cows, respectively). Thus, on balance, considering all currently available evidence, the majority of studies, and the most definitive and comprehensive studies, clearly demonstrate a significant increase in IGF-I concentrations in milk from rbST-treated cows.
The US Food and Drug Administration reviewed most of the same studies and concluded that rbST use does lead to statistically significant increases in the IGF-I levels in milk (Juskevich and Guyer, 1990; FDA, 1993).
Another study on the effect of rbST on IGF-I levels in milk, which was reviewed by neither JECFA nor the US FDA, found an even larger increase in IGF-I levels in the milk of rbST-treated cows (Prosser et al., 1989). The study involved rbST treatment to six cows near the end of the lactation cycle, when IGF-I levels are normally at a minimum. At the end of one week of daily rbST injections, the milk of rbST-treated cows had IGF-I levels at least 3.6 times the levels in the milk of untreated cows. The levels were still rising at the end of the treatment period. How high the levels would have gone if the rbST injections had continued is not known.
Does IGF-I survive digestion?
If levels of IGF-I increase in the milk of rbST-treated cows, the question of whether IGF-I survives digestion in the stomach is important.
New data, as well as earlier studies not considered by JECFA in 1992, suggest that IGF-I survives digestion in vivo. Three studies involving neonate rats or calves suggest, either directly or indirectly, that IGF-I does survive digestion and remain bioactive. The first study involved orally administering labeled 125I-IGF-I to suckling rats and found that more than three quarters (78%) of the labeled 125I-IGF-I was retained in the stomach and intestinal lining, where the authors proposed that it could have a local effect (Phillips et al., 1990). The second study involved orally administering labeled 125I-IGF-I to calves. It found a small amount of 125I-IGF-I in the circulatory system, indicating that it not only survived digestion, but was also taken up into the blood (Baumrucker et al., 1992).
The third study investigated the effect of feeding calves for seven days with either bovine milk replacer (which lacks IGF-I) alone or supplemented with 750 micrograms/liter of IGF-I (Baumrucker and Blum, 1993). In the first three days no differences in serum IGF-I levels were observed between the two diets. However, the milk replacer with added IGF-I did lead to a transient decrease in serum insulin (within 2 hours), a transient increase in serum prolactin levels (within 4-8 hours), increased DNA synthesis (as measured by thymidine incorporation) in jejunal and ileal intestinal explants, and an increase in number of IGF-I receptors in jejunal and ileal microsomal membranes (which may explain the increased DNA synthesis). These effects suggest indirectly that IGF-I did survive digestion, was active in the lower gastrointestinal tract, and was to some degree absorbed into the blood. Collectively, these three studies suggest that orally administered IGF-I at least partially survives digestion, binds to IGF-I receptors in cells lining the GI tract, stimulates synthesis of its own receptor, stimulates cellular proliferation, and is absorbed into the blood where it can affect levels of other hormones.
A new study, published in 1995, provides both clear evidence that IGF-I survives digestion (Xian et al, 1995) and an explanation for why the oral IGF-I feeding studies looked at by JECFA in 1992 had ambiguous results, as is discussed below. Both the Monsanto-sponsored and the Elanco-sponsored studies previously considered by JECFA involved feeding free rIGF-I by itself to rats. Neither used IGF-I associated with its binding proteins (IGFBPs). IGFBPs are resistant to acidic conditions and may enable IGF-I to survive digestion in the stomach (Corps and Brown, 1987; Donovan and Odle, 1994). Furthermore, in these two previous studies, the free IGF-I was not mixed with other constituents of milk, such as casein, which more recent evidence indicates protect it from digestion.
On theoretical grounds alone, one might expect IGF-I to survive digestion. Milk has recently been shown to contain a number of growth factors, including ones that stimulate growth of the gut (Donovan and Odle, 1994). Since newborns and young infants have the fastest growth rates, one would expect that any growth factors that a mother might give her infant would be found at their highest concentrations in the earliest milk in the lactation, when the infant is growing the fastest. Furthermore, for the mother’s milk to be able to deliver growth factors that will affect the gastrointestinal tract, those growth factors must survive digestion in the stomach and reach the upper and lower intestines where they can have local stimulatory/growth effects.
Both IGF-I and epidermal growth factor (EGF), a protein growth hormone related to IGF-I, are known to stimulate intestinal epithelial growth in vitro (Corps and Brown, 1987). IGF-I receptors are found on the intestinal epithelium of rats (Laburthe et al., 1988) which suggests that orally administered IGFs may exert mitogenic responses in the gut, especially in newborn animals (Koldovsky et al., 1992). In addition, both IGF-I and EGF occur in both bovine and human milk, with the level highest at the start of the lactational cycle. Particularly high concentrations are found in the colostrum and early milk, which is just when the infant gut is doing most of its growing, suggesting that IGF-I performs a physiological function immediately after birth, promoting development of the gut. Indeed, growth of the gut in infant animals appears to be due in part to the presence of EGF and other growth factors in the colostrum and milk.
Since EGF has long been known to survive digestion (Thornburg et al., 1984), some authors have inferred that IGF-I probably also survives digestion (Corps and Brown, 1987). A study published in 1993 showed that EGF survives digestion through the protective effects of the major milk protein casein (Playford et al., 1993). This suggests that the same mechanism might occur with IGF-I.
And this is just what a 1995 study found. The study was designed "to investigate the potential of IGF-I peptides as therapeutics in the gut" (Xian et al., 1995: 215). By therapeutics, the authors meant the use of IGF-I to stimulate growth of the gut, which tends to waste away in hospital patients on parenteral nutrition who eat little solid food. The study involved feeding human IGF-I to suckling rats in the presence of a number of proteins, such as bovine casein, IGF-binding protein-3, bovine serum albumin (BSA), lactoferrin and an antibody to IGF-I. The study found that free IGF-I orally administered to rats was quickly digested in the stomach. However, IGF-I in the presence of casein easily survived digestion in the stomach and made its way to the intestine: "In stomach, casein was the most effective protein, with near complete inhibition of IGF-I degradation at casein concentrations of 10 mg/ml or higher . . . All three proteins [BSA, casein, and lactoferrin] were less protective in the duodenal flushings than in the stomach flushings. Nevertheless, casein remained the most effective protein, with 40 mg/ml conferring maximum protection, at which IGF-I remained 80% intact by TCA" (Xian, et al., 1995: 221).
Since casein levels in bovine milk average 25-40 mg/ml, the experiment suggests that bovine milk has enough casein to partially or fully protect IGF-I from digestion in the stomach, enabling it to pass into the small and large intestine, where it might have a local stimulatory effect on epithelial cells. Indeed, the authors of this study concluded that using casein may make it possible to give therapeutic oral doses of IGF-I: "It can be concluded that IGF-I cannot be expected to retain bioactivity if delivered orally because of rapid proteolysis in the upper gut, but the use of IGF antibodies and casein could represent useful approaches for IGF-I protection in oral formulae" (Xian et al., 1995: 215).
The demonstration that IGF-I survives digestion through the protective effects of casein makes irrelevant the argument that human saliva contains IGF-I at levels greater than the quantities that would be consumed in milk. As the IGF-I produced by salivary glands is free IGF-I, without the protective effect of casein, it is unlikely to survive digestion.
Two earlier oral feeding studiesone sponsored by Monsanto and one by Elancoconcluded that IGF-I does not survive digestion. Those studies are not definitive because rats were given free IGF-I, without casein or other protective proteins. Thus one would not expect the IGF-I to survive digestion in these studies. Even so, a careful review of the Monsanto study suggested that some small amount of the IGF-I administered in this study survived digestion and affected the rats’ growth rate (Hansen, 1993).
In sum, we feel that the data clearly show that IGF-I survives digestion in animal studies, makes its way into the gastrointestinal tract, and has a mitogenic (i.e. promotes cell division) effect on cells in the gastrointestinal tract. Although no data are available on humans, there is ample reason to expect that we are like other mammals in this regard.
What are the possible adverse health effects of increased exposure to IGF-I in milk?
If IGF-I survives digestion and if IGF-I levels are increased in milk from rbST-treated cows, it is important to examine the potential adverse effects of increased exposure to IGF-I on humans. Scientific understanding of how IGF-I works and its potential health impacts in humans has grown considerably in the more than five years since JECFA met to discuss rbST. The most important potential adverse effects of IGF-I arise from the fact that it is a potent mitogen for a number of cell types and has been associated with the growth of numerous tumors, including colon (Tricoli et al., 1986), breast (Rosen et al., 1991; Lippman, 1991), smooth muscle (Hoppener et al., 1988) and others (Pavelic et al., 1986).
Most of the recent advances in the knowledge in this area were discussed at a 1995 National Institutes of Health (NIH) Conference on the insulin-like growth factor and cancer, and summarized in the Annals of Internal Medicine in 1995. The advance in knowledge from the NIH 1990 Technical Assessment Conference on BST, which suggested further study on the local effects of IGF-I on the gastroin-testinal tract, to the 1995 Conference, is quite striking.
The 1995 NIH Conference focused on the role that the insulin-like growth factor (IGF) system plays in the development and spread of cancer. The IGF system consists of three growth factors (IGF-I, IGF-II, and insulin), a number of binding proteins (at least 6 characterized so far), and receptors for each of the growth factors. It is clear that IGFs (e.g. IGF-I and IGF-II) are needed for normal growth and develop-ment. However, it is now becoming evident that IGFs may also play an important part in what goes awry in the development and spread of many cancers. The summary of the NIH conference concisely states this:
"As could be predicted from the importance of IGFs, their binding proteins, and their receptors in normal cellular growth and development, it has become apparent over the past few years that IGFs are important mitogens in many types of malignancies. Although these conclusions were initially derived from in vitro studies, IGFs may enhance in vivo tumor cell formation, growth and even metastasis. Insulin-like growth factors may reach tumors either from the circulation (endocrine) or as a result of local production by the tumor itself (autocrine) or by adjacent stromal tissue (paracrine). Tumors also express many of the IGF-binding proteins, which modulate IGF action, and IGF receptors, which mediate the effects of IGFs on tumors" (LeRoith, 1995: 54)
Not surprisingly, most of the cancers that IGF-I is associated with occur in tissues where IGF-I normally plays an important growth role, including the mammary, cardiovascular, respiratory, and nervous systems, the skeleton, and the intestinal tract. We will briefly describe the evidence for the association in each of these systems.
IGF-I appears to play a strong role in breast cancer. Not only do breast cancer cells react strongly (i.e., grow and divide) in the presence of low levels of IGF-I, but the tissue surrounding the breast cancer cells produces IGF-I. Some strategies for combating breast cancer involve reducing circulating levels of IGF-I. As summa-rized at the NIH conference by Dr. LeRoith:
"IGFs have been shown to be involved in breast cancer. . . . Breast cancer cells in vivo express low levels of IGF-II, whereas the adjacent stromal tissue expresses IGF-I. . . . Estrogen receptor-positive tumors will thus respond to antiestrogens such as tamoxifen, which is widely used clinically. Initially, it was thought to affect cancer cells primarily by blocking the activation of estrogen receptors; it has also been shown, however, to decrease circulating IGF-I levels in women with breast cancer and may thus prove effective in treating both estrogen receptor-positive and estrogen receptor-negative cancers. Another agent that inhibits the proliferation of breast cancer cells is retinoic acid and its derivatives. . . . Like tamoxifen, however, retinoic acid may also reduce circulating IGF-I levels and may thus affect tumor growth in vivo by more than one mechanism. The above data suggest that IGFs are likely to be involved in breast cancer at the level of tumor growth and perhaps at the level of initial development and later metastases. Ongoing studies involve attempts to interfere with the IGF system to develop additional therapeutic regimens" (Baserga, 1995: 55-56).
In the skeletal system, IGF-I has been associated with osteosarcoma. The tumor seems to strike children with the most rapidly growing bones, and has been shown in vitro to respond to IGF-I (i.e. grow and divide in its presence and grow more slowly in its absence). Suggested therapies include trying to reduce IGF-I levels. As summarized at the NIH conference:
"Osteosarcoma is the most common bone tumor in children, usually occurring during the adolescent growth spurt at sites of rapid bone growth. Because IGF-I was initially described as the factor produced that directly mediated the effect of growth hormone on skeletal growth, there has been interest in a potential role of IGF-I in the pathogenesis of osteosarcoma. Support for a role for IGF-I in osteosarcoma growth comes from data showing that IGF-I is a potent mitogen for human osteogenic sarcoma cells. Further, several reports have shown that a rat chondrosarcoma (a closely related tumor) and a murine osteosarcoma are growth inhibited in animals that have a hypophysectomy [so they no longer produce GH], presumably through the inhibition of the growth hormone-IGF-I axis. . . . It therefore appears that the growth hormone-IGF-I axis may play a role in the unregulated proliferation of osteosarcoma tumor cells and that blocking this axis using somatostatin analogs that reduce circulating levels of growth hormone and IGF-I may have therapeutic potential" (Helman, 1995: 57).
In the respiratory system, IGF-I is known to play a role in normal lung development and has also been implicated in lung cancer. As pointed out in a 1992 British study, "IGFs seem important in lung development, and are also implicated in growth regulation of lung tumors. Primary lung tumors possess IGF-I binding sites as shown by autoradiography, with the highest density of receptors in squamous cancers and small cell lung cancer. . . . Thus there is good evidence that lung cancer cells produce IGF-I and IGF BPs, express IGF binding sites and exhibit a mitogenic response to exogenous IGF-I, suggesting that IGF-I can function as an autocrine [tumor-produced] growth factor for lung cancer" (Macaulay, 1992: 312).
In the circulatory system, IGF-I has been shown to have angiogenic properties (i.e., to promote growth of blood vessels). These properties are important to tumors as blood vessels supply food and oxygen to cancerous tissues. Getting nutrients and oxygen to the center of large tumor is difficult, and some tumors secrete growth factors to promote angiogenesis.
Excessive angiogenisis can be dangerous by itself, i.e. in the absence of a tumor. In fact, a recent study in mice found that retinal neovascularization (excessive growth of tiny blood vessels on the retina, which blocks vision), a major cause of untreatable blindness, is inversely related to growth hormone and IGF-I levels, and suggested that depressing IGF-I levels could be a potentially important therapy to protect against neovascularization. As the authors state, "Retinal neovascularization was inhibited in these mice in inverse proportion to serum levels of GH and a downstream effector, insulin-like growth factor-I (IGF-I). Inhibition was reversed with exogenous IGF-I administration. . . . These data suggest that systemic inhibition of GH or IGF-I, or both, may have therapeutic potential in preventing some forms of retinopathy" (Smith et al., 1997: 1706).
The potential effects of IGF-I on the gastrointestinal system are of special concern. A 1987 study found that IGF-I stimulates growth of intestinal epithelial cells, a sign that these cells are programmed to respond to IGF-I (Corps and Brown, 1987). The authors noted that epidermal growth factor (EGF), a protein growth hormone related to IGF-I, has been shown to pass intact through the stomach to the small intestine where it is absorbed into the bloodstream, and wondered whether the same would be true for IGF-I. In part because of these concerns and in part because they felt that the data showed that IGF-I levels are statistically significantly increased in milk from rbST-treated cows, the 1990 US National Institutes of Health (NIH) Technical Assessment Conference on Recombinant Bovine Somatotropin concluded, "Whether the additional amounts of IGF-I in milk from [rbST-treated] cows has a local effect in the esophagus, stomach or intestines is unknown." One of the six recommendations for further research was, "Determine the acute and chronic action of IGF-I if any, in the upper gastrointestinal tract" (NIH, 1991).
More recent studies have suggested that IGF-I does have growth effects on the gut. In 1988 French and Danish researchers showed that IGF-I receptors are found throughout the intestines, with the highest density occurring in the crypt cells in the epithelium of the colon: "125I-IGF-I and 125I-IGF-II binding is 4.0 and 1.8-fold higher in crypt cells than in villus cells, respectively. Specific 125I-IGF-I binding is detectable throughout the gastrointestinal tract. The level of IGF binding is similar in stomach, small intestine, and cecum, but higher values are observed in the colon" (Laburthe et al., 1988: G457).
A 1992 study not reviewed by JECFA found that intraluminal infusion of IGF-I in rats at concentrations equivalent to those found in bovine milk increased the cellularity of the intestinal mucosa (Olanrewaju et al., 1992). A 1994 study found that epithelial crypt cells in healthy human duodenal tissue proliferated at twice the normal rate when exposed to recombinant human IGF-I at concentrations of 400ng/ml, while "lower concentrations (100 and 200 ng/mL) also increased crypt epithelial cells in preliminary dose-response studies" (Challacombe and Wheeler, 1994: 816). Although the levels of IGF-I used were 100 ng/ml or higher, the main author, Dr. David Challacombe, of the Somerset Children’s Research Unit or Taunton and Somerset Hospital, urged further study, stating of his study that, "It could mean that if you have higher levels of IGF-I in BST-treated milk, it could increase cell proliferation in the small bowel, and there’s always the possibility they could form abnormally into a tumour of some kind" (Coghlan, 1994: 15).
Further evidence supporting this possibility is the finding that 5 of 8 human colorectal cancer cell lines were responsive to IGF-I (Lahm et al., 1992). These cell lines were exquisitely sensitive to IGF-I, with 30 ng/ml tripling cancer cell growth and with 1.9 – 6.5 ng/mllevels similar to those found in bovine milkincreasing cancer cell growth 1.5-fold: "At 30 ng ml-1 both factors [IGF-I and IGF-II] enhanced growth up to 3-fold. They induced half maximal stimulation at 1.9 – 6.51 ng ml-1" (Lahm et al., 1992: 341). Another study noted that "Immunoreactive IGF-1 has also been shown to be increased in primary human lung and colon carcinomas compared with adjacent normal tissue. Specific IGF-1 receptors have also been characterized on human T-lymphoblasts, neurogliomas, and colon carcinomas" (Ezzat and Melmed, 1991). Furthermore, IGF-I mRNA has been found in colon carcinoma, suggesting that these tumors produce IGF-I to stimulate their own growth (Tricoli et al., 1986).
The lines of evidence cited aboveincreased level of IGF-I receptors, the sensitivity of colon carcinomas to IGF-I, and production of IGF-I by the carcinoma itself support the hypothesis that the colon could be at special risk from increased IGF-I levels. More support for this hypothesis comes from studies on acromegaly, a disease in which patients have significantly elevated endogenous levels of total and free serum IGF-I (Juul et al., 1994). Several studies have found acromegaliacs are at increased risk of colon tumors and precancerous colonic polyps. A recent review concluded that, now that acromegaliacs are living to older ages, physicians "need to remain aware of the potentially deleterious long-term consequences of previous GH excess and, in this context, the possibility of malignant transformation of the colonic polyps seems paramount" (Tremble and McGregor, 1994: 10). In addition to cancer per se, colon polyps, especially adenomatous polyps, are important as it is "generally established that colonic adenomatous polyps are premalignant lesions that have the potential to develop into adenocarcinoma of the colon" (Klein et al., 1982).
An early study found that among 12 patients with acromegaly, 3 had colon carcinoma and 2 had adenomatous polyps of the colon (Ituarte et al., 1984). Since the expected number of colon cancers in a 12 person sample is less than one, the finding of 3 was very highly statistically significant (p < 0.001). A study from 1982 of 17 patients with acromegaly found that 9 had colonic polyps (Klein et al., 1982). Polyps were removed from 8 of the patients. In 5 patients, the polyps were adenomatous and in four of the five there were multiple polyps. In addition, during the course of that study, the authors "identified four cases of colon cancer in a total of 43 patients with acromegaly" and concluded that their "study identifies a unique group of patients that are at risk of the development of colonic polyps and perhaps colon cancer" (Klein et al., 1982: 29).
One retrospective study of gastrointestinal tumors in 48 acromegalic patients found a statistically significant standardized incidence ratio (SIR) of 4.6 for all GI cancers and 6.1 for colorectal cancers; i.e., GI cancers were 4.6 times as prevalent and colorectal cancers 6.1 times as prevalent among acromegaliacs as among the general population (Pines et al., 1985). Another study of 52 patients with acromegaly found a prevalence ratio for colorectal cancer of 6.9 per 100, which is considered quite high, with the authors concluding that "this and most other recent investigations have observed a trend of increased risk for colon cancer and polyps among patients with acromegaly" (Brunner et al., 1990: 70). Finally, a prospective study of 23 patients with acromegaly found that 8 (35%) had premalignant adenomatous colon polyps, which "exceeds the 12% (P < 0.01) frequency of polyps noted in normal persons" (Ezzat et al., 1991: 754).
Discussion
Our focal concern is that increased levels of IGF-I in milk, by stimulating growth of intestinal cells, could increase the risk of colon cancer, the third most common cause of cancer mortality in the US (Devesa et al., 1995).
The evidence seems sufficient to conclude that IGF-I is both a paracrine and autocrine growth factor for colon cancers (Tricoli et al., 1986, Lahm et al., 1992) This means it is appropriate to focus on local levels of IGF-I in the intestine, rather than on levels of IGF-I in the bloodstream. One study of human colorectal cancer cell lines found that IGF-I made tumor cells grow 1.5-fold faster at IGF-I levels (e.g. 1.9 – 6.51 ng ml-1) similar to those found in bovine milk. Thus, an increase in average levels of IGF-I in milk, which seems likely to occur with widespread use of rbST, could in theory increase human colon cancer risks. We do not believe enough evidence exists currently to assess this potential risk quantitatively. In particular, assessing patterns of public exposure to increased IGF-I in milk is an extremely complex process, requiring many assumptions. However, this subject seems to demand intensive further research toward the goals of further elucidating and quantifying the risk.
With no basis for quantitative risk assessment, there are nevertheless several principles that can serve as guides for inferences on this question. If a large increase in IGF-I exposure in a small population (i.e. acromegalics) notably increases cancer risk, then it is plausible that a small increase in IGF-I for a much larger population could pose a significant public health risk. When the substance of concern is present in milk, as is IGF-I, exposure will be widespread. In Western countries, cow’s milk is consumed by virtually the entire population in childhood, and by much of the population for a whole lifetime. Exposure during infancy and childhood raises some additional concerns, because these age groups drink the largest amount of milk on a body weight basis, and because growth and development are most rapid during childhood. Thus, higher exposures to IGF-I in childhood could set life-long processes in motion that determine later risks.
Other authors, particularly Samuel Epstein, have argued that an increase in IGF-I in milk could increase the risk of breast cancer as well (Epstein, 1996).
2. Does use of rbST increase mastitis in cows and lead to increased antibiotic use, contributing to drug resistance in bacteria and residues in dairy products?
A second important concern is that rbST use increases disease rates, especially mastitis rates, in treated cows, thereby increasing drug use to treat those diseases, which, in turn, can contribute to increased antibiotic resistance in bacteria and to an increased likelihood of residues in milk and meat. The crucial evidence on this issue falls into four areas: 1. Does use of rbST increase mastitis rates in cows? 2. Are cases of mastitis more severe/less easy to control in cows treated with rbST compared to controls? 3. Does rbST use increase the amounts of drugs given to cattle to treat diseases? 4. Does increased drug use in dairy cattle exacerbate the problem of antibiotic resistance in pathogenic bacteria and/or residues in milk? Much of the data analysis that follows is based on the work of David Kronfeld, who has followed this issue very carefully in the U.S. and has published some major analyses of these data (Kronfeld, 1994, 1997).
Does use of rbST increase mastitis rates in cows?
Most of the evidence needed to answer this question has emerged since 1992. According to the U.S. Food and Drug Administration, which used data from eight Monsanto-sponsored trials in its decision in 1993 to approve Monsanto’s rbST product (POSILAC), the answer is yes. The data from these eight trials, which involved 487 cows, showed that during the period of rbST treatment, mastitis incidence increased by 76% in primiparous cows (from 21 cases to 37 cases per 100 cows, for control and rbST-treated cows, respectively; p = 0.015), and by 50% for multiparous cows (from 36 cases to 54 cases per 100 cows, for control and rbST-treated cows, respectively; p = 0.002) (See Table 2). Overall, the increase was 53% (from 32 cases to 49 cases per 100 cows, for control and rbST-treated cows, respectively; p = 0.0001) (FDA, 1993, Kronfeld, 1997).
The approval by US FDA of POSILAC, was conditioned, in part, on the establishment of a post-approval monitoring program (PAMP). The PAMP included monitoring cows from 28 herds for a variety of health problems as well as accumulating data on violative drug residues in milk. The data from the PAMP (see Table 2), which involved 1128 cows, showed that during the period of rbST treatment, mastitis incidence increased by 22% in primiparous cows (from 27 to 33 cases per 100 cows, for control and rbST-treated cows, respectively; p = 0.17, i.e., not statistically significant), and by 34% in multiparous cows (from 44 to 59 cases per 100 cows, for control and rbST-treated cows, respectively; p = 0.0001). Overall, the increase was 32% (from 37 to 49 cases per 100 cows, for control and rbST-treated cows, respectively; p = 0.0001) (Kronfeld, 1997). The large differences in statistical significance between the results from primiparous and multiparous cows can be explained by the fact that the number of primiparous cows was much smaller.
In addition to these newer studies carried out for FDA, two earlier studies, both published in 1991 (and involving some of the same data that FDA looked at), found an increase in mastitis incidence in rbST-treated cows compared to controls. One study looked at 15 commercial herds and found an increase of 47%, which was not statistically significant (p = .097) (Thomas et al., 1991), while the other looked at 14 herds and found an increase of 35%, which was highly statistically significant (p < 0.01) (Craven, 1991).
Kronfeld has analyzed the results of the various mastitis studies, especially the 8 pre-approval trials and the data on the 28 herds that were part of the PAMP, and has pointed out a number of things about rbST-associated mastitis (Kronfeld, 1994, 1997). First, the effect of rbST on mastitis is variable (or "inconsistent" in Kronfeld’s terminology), with an increased frequency of mastitis being observed in only one-half to one-third of the rbST-treated herds. Thus, in the 8 pre-approval herds, while there was an overall increase in mastitis of 76% for the primiparous cows, this increase was actually composed of no increase in 4 herds and a 152% average increase in the other 4 herds. In the study of 15 commercial herds that found an increase in mastitis incidence of 47% overall, there actually was a 103% increase in 7 affected herds and little or no increase (or a slight decrease) in the other 8 herds. This variability of mastitis effect means that the global averages hide the fact that some (one-third to one-half) herds are hit heavily by mastitis (i.e. it more than doubles), while other herds are not hit hard at all. Thus, the pooling of data or focusing on averages can obscure the seriousness of mastitis that occurs in some rbST-treated herds.
Are cases of mastitis more severe/less easy to control in cows treated with rbST compared to controls?
RbST-associated mastitis appears to be harder to treat than "normal" mastitis. In one trial from Vermont, the average length of treatment for a case of mastitis was almost six times longer in the rbST-treated cows compared to untreated cows (8.9 days vs. 1.5 days); the authors attribute the greater length of treatment to infection with Staphylcoccus aureus in the rbST-treated cows. S. aureus is associated with particularly difficult cases of mastitis (Pell et al., 1992). Data from a technical manual on rbST distributed to veterinarians by Monsanto, and based on 10 U.S. rbST-trials (and including all 8 trials that the US FDA used to grant approval), showed that the percentage of bacterial isolates from clinical mastitis cases that contained S. aureus increased by 62%, from 11.1% in controls to 18.0% in rbST- treated cows (Monsanto, 1993).
In one of the original eight trials in the U.S., there was extensive extra-label use of antibiotics not approved for use in dairy cattle to treat mastitis, suggesting that the legal drugs (such as penicillin) were relatively ineffective (Kronfeld, 1997). In another study, the percent of mastitis cases harboring antibiotic-resistant bacteria was one-third higher in the rbST-treated cows compared to controls (65% and 49%, respectively) (Kronfeld, 1997).
Does rbST use increase the amounts of drugs given to cattle to treat diseases?
Both increased incidence of mastitis and more severe or longer-lasting cases of mastitis can lead to greater antibiotic use. In the Vermont study cited above, there were more than seven times as many cases of mastitis in rbST-treated cows compared to controls (29 vs. 4), while the average length of antibiotic treatment was almost six times as long (8.9 days vs. 1.5 days), leading to a 43-fold increase in the total duration of antibiotic treatment for rbST-treated cows, compared to controls (Pell et al., 1992). In the study of 15 commercial herds that found a 47% overall increase in mastitis in rbST-treated cows, antibiotic treatment doubled in rbST-treated cows compared to controls. If we look only at the 7 herds which had increased mastitis incidence, then total duration of antibiotic treatment was 2.7 times as high in rbST-treated cows compared to controls (Kronfeld, 1997). In the PAMP trial, which consisted of 28 herds and 1128 animals, total duration of antibiotic treatment for mastitis was 2.3 times as high in primiparous rbST-treated cows compared to controls, and 1.3 times as high, rbST-treated vs. controls, in multiparous cows; both effects were highly statistically significant (P < 0.01).
Finally, as a condition of POSILAC® approval in the U.S., the FDA required that Monsanto include a package insert which explicitly states that it will increase drug use: "Use of POSILAC is associated with increased frequency of use of medication in cows for mastitis and other health problems."
Does increased drug use in dairy cattle exacerbate the problems of antibiotic resistance in pathogenic bacteria and residues in milk?
Another part of the PAMP consisted of looking at the national figures for violative drug residues in milk. These data were presented at a meeting of the FDA’s Veterinary Medicine Advisory Committee (VMAC) in 1996. As Dr. Kronfeld notes, violative antibiotic residues in milk tankers increased from 0.05% in 1992 and 1993, before rbST (sic) use, to 0.06% in 1994 and 0.09% in 1995. If the 1.8-fold overall increase from 0.05 to 0.09% were confined to the 10% of herds using rbST, then an 8-fold increase in milk violations would be occurring in these herds (Kronfeld, 1997: 164-165).
The US figures on violative antibiotic residues probably understate the true incidence of residues. Bulk tanks of milk in the US are routinely tested for residues of antibiotics in the beta lactam family (which includes penicillin) and any milk found with violative residues is discarded. However, many more antibiotics are used on dairy cows. Under U.S. law, any drug approved for any use on humans or animals, with just a handful of exceptions, can be used on dairy cows, if used under a veterinarian’s supervision. The FDA spot checks 500 samples per year for 12 drugs, but this testing seems likely to miss many drugs in use. Good, accurate tests that reliably detect low-level residues simply do not exist for the bulk of the drugs in use in dairy cows in the US. Thus, as the US General Accounting Office (a watchdog arm of Congress) pointed out in 1990 and 1992 (GAO, 1990, 1992), the existing antibiotic testing program cannot guarantee that illegal residues are not present in the milk supply. This situation has not drastically improved in the last five years.
Since data were not taken on the antibiotic residue levels from the milk of treated and untreated cows, either in the 28 herds involved in the PAMP or in the 8 herds used to gain approval in the U.S., there is no direct evidence on antibiotic residues in milk from rbST-treated cows. However, the studies cited above clearly show that mastitis rates do increase, and use of drugs to treat mastitis increases.
Greater use of antibiotics in dairy cattle is of concern not just because of residues, which some authorities believe may cause adverse (i.e. allergic) reactions in a few sensitive individuals, but also because it contributes to the growth of antibiotic resistance in bacteria, an important public health problem. Resistance can initially develop in both pathogens and harmless bacteria in treated animals, and later be transferred to disease-causing bacteria that infect humans, with the end result that a given antibiotic may not be effective in treating disease.
In general, use of antibiotics contributes to the antibiotic resistance problem by selecting for bacteria that are resistant to the given antibiotic. Any factors that tend to increase antibiotic use can contribute to or exacerbate this problem. The most effective way to prevent and/or delay resistance is to use the drug as selectively as possible. When resistance is present, however, control of infections may require switching to other antibiotics, which can be effective in the short term but may also contribute to development of strains of bacteria that are resistant to multiple drugs.
Antibiotic resistance is carried in bacterial genes called R factors, which code for proteins that prevent specific antibiotics from having toxic actions on the cell. R factors may be located on the main chromosome(s) of bacteria, or on small genetic elements called plasmids. R factors on the main chromosome are not very mobile and can be transferred to other similar bacteria only through conjugation, a sexual form of bacterial reproduction, or through the fusion of different mating types. Plasmids, unlike chromosomes, are highly mobile elements that can easily be transferred among bacteria of all types, i.e. they can move between bacteria of different genera or different families. Indeed, plasmids can even move from dead bacteria into living bacteria. Furthermore, as plasmids move from bacteria to bacteria they can accumulate R factors for resistance to a number of different antibiotics on the same plasmid. Plasmid-borne resistance is therefore a far more serious concern than chromosomal resistance.
Antibiotic use in dairy cows can lead to resistance that could potentially affect human health by several pathways. The most direct path is if a resistant pathogen becomes established in dairy cows, either because competing non-resistant bacteria in the cow are decimated by repeated antibiotic use or because R-factors are passed from non-pathogenic resistant bacteria in the cow. The pathogen could then be present in meat or milk, which could produce human exposure to infection that was resistant to antibiotics.
An indirect path also could exist if non-pathogenic bacteria with an R factor were present in meat and/or milk. The R-factor-containing plasmid could then move from non-pathogenic bacteria to pathogenic ones in the human gut, creating the potential for a difficult-to-treat infection. An even more indirect path would be for antibiotic residues in milk to select for resistance in bacteria in the intestine.
In the past, it has been argued that the antibiotic levels in milk are far too low to select for antibiotic resistance. However, a paper published in 1993 demonstrated that even FDA "safe levels" (from 10 to 150 parts per billion) of antibiotic residues in milk can select for disease resistance in Staphylococcus aureus (Brady et al., 1993). In addition to its role in severe cases of mastitis, as noted above, S. aureus is responsible for many serious human infections in hospitals. The authors found that residues of one antibiotic in the milk increased the rate at which S. aureus evolved resistance to that antibiotic by 600 percent; with residues of three antibiotics, the increase was 2,700 percent.
Data from a technical manual on rbST distributed to veterinarians by Monsanto, and based on 10 U.S. rbST-trials (and including all 8 trials that the US FDA used to grant approval), showed that the percentage of bacterial isolates from clinical mastitis cases that contained S. aureus increased by 62% in rbST-treated cows (Monsanto, 1994). The authors of the S. aureus study concluded "that greater emphasis should be placed on keeping the milk supply residue free rather than reliance on maintaining the working residue levels suggested by the term ‘safe levels’ " (Brady et al., 1993: 232).
In summary, convincing evidence from both pre-approval controlled trials of rbST and from the PAMP in the US clearly indicates that rbST use can dramatically increase mastitis rates in some treated herds, requiring increased use of antibiotics. The additional antibiotic use due to rbST use cannot help but contribute to the general problem of antibiotic resistance in pathogenic bacteria. In addition, there is some evidence (and an obvious need for further research) to indicate that antibiotic residues in milk are capable under some conditions of selecting for resistant bacteria in the human intestinal tract. To the extent that rbST use would lead to increased occurrence of antibiotic residues in milk, which it appears it has done in the U.S., widespread use of rbST could exacerbate the resistance problem by this pathway as well.
Part 1 – Report
Part
1b – Report Continued
Part
2 – References
Part 3 – Tables