Cyclophosphamide induces caspase 9-dependent apoptosis in 9L tumor cells.

Cyclophosphamide (CPA), a widely used oxazaphosphorine anti-cancer prodrug, is inactive until it is metabolized by cytochrome P450 to yield phosphoramide mustard and acrolein, which alkylate DNA and proteins, respectively. Tumor cells transduced with the human cytochrome P450 gene CYP2B6 are greatly sensitized to CPA, however, the pathway of CPA-induced cell death is unknown. The present study investigates the cytotoxic events induced by CPA in 9L gliosarcoma cells retrovirally transduced with CYP2B6, or induced in wild-type 9L cells treated with mafosfamide (MFA) or 4-hydroperoxyifosfamide (4OOH-IFA), chemically activated forms of CPA and its isomer ifosfamide. CPA and MFA were both shown to effect tumor cell death by stimulating apoptosis, as evidenced by the induction of plasma membrane blebbing, DNA fragmentation, and cleavage of the caspase 3 and caspase 7 substrate poly(ADP-ribose) polymerase (PARP) in drug-treated cells. Caspase 9 was identified as the regulatory upstream caspase activated in 9L cells treated with CPA, MFA, or 4OOH-IFA, implicating the mitochondrial apoptotic pathway in oxazaphosphorine-induced tumor cell death. Correspondingly, expression of the mitochondrial proapoptotic factor Bax enhanced caspase 9 activation, plasma membrane blebbing, and drug-induced cytotoxicity. Conversely, overexpression of the mitochondrial antiapoptotic factor Bcl-2 blocked caspase 9 activation, leading to an inhibition of drug-induced plasma membrane permeability and blebbing, terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity, PARP cleavage, Annexin V positivity, and drug-induced cell death. Although Bcl-2 thus blocked the cytotoxic effects of activated CPA, it did not inhibit the drug's cytostatic effects. CPA induced S-phase cell cycle arrest followed by conversion to an apoptotic pre-G1 state in wild-type 9L cells; by contrast, Bcl-2-expressing 9L cells accumulated in G2/M in response to CPA treatment. Intratumoral expression of Bcl-2 and related family members, including both apoptotic and antiapoptotic factors, is thus an important determinant of the responsiveness of tumor cells to CPA and ifosfamide, both in the context of conventional chemotherapy and in patients sensitized to these oxazaphosphorine drugs by the use of cytochrome P450-based gene therapy.

Update on migration research and regulatory initiatives.

The Food and Drug Administration's (FDA) current research programme on migration from food packaging materials focuses on high-temperature packaging applications. Results from two studies conducted by Arthur D. Little, Inc., under contract to the FDA, are presented as well as highlights of the FDA's research programme on migration from microwave heat susceptor packaging. Some regulatory policy issues affecting the FDA's future regulation of food packaging materials are also examined.

Migration of BHT and Irganox 1010 from low-density polyethylene (LDPE) to foods and food-simulating liquids.

The most widely used food-wrapping material is low-density polyethylene (LDPE). Food-wrap grades contain antioxidants to minimize degradation during processing and, in the final films, such additives are normally present at levels of several hundred ppm. During use, the antioxidants may migrate into food stored in LDPE wraps. Two typical antioxidants, BHT and Irganox 1010, were radiolabelled to allow accurate analytical measurement of the extent of their migration into foods and food-simulating liquids (FSL). The results show that BHT, a much smaller and more volatile molecule than Irganox 1010, migrates more rapidly into foods, but the differences are less for FSL. In most instances, migration appears to be controlled by diffusion of the antioxidant in the polymer, and the quantity lost can be correlated in a linear fashion with the square root of time. With aqueous FSL, and, presumably aqueous-type foods, however, anomalies result; the migration is often erratic, but is more closely related to time than to the square root of time. A tentative model developed to explain these facts assumes that the antioxidants decompose in aqueous media and the net migration rate is controlled largely by the rate of chemical decomposition. It is also shown that dry foods can be surprisingly effective sinks for antioxidants under typical storage conditions.

Migration of Irganox 1010 from ethylene-vinyl acetate films to foods and food-simulating liquids.

In a series of experiments on the migration of the antioxidant Irganox 1010 from ethylene-vinyl acetate (EVA) films into food-simulating liquids and foods, the antioxidant was found to migrate rapidly from EVA film into n-heptane, 100% ethanol and corn oil. The rate of migration into these media was greater from EVA than from low-density polyethylene (LDPE) under comparable conditions. In contrast, little migration of Irganox 1010 was recorded on exposure of the EVA film to aqueous media, whereas migration from LDPE into such media was relatively high.

Indirect food additive migration from polymeric food packaging materials.

Many foods contact polymeric packaging materials which contain residues of the polymerization process or additives employed to facilitate processing. The extent of migration of such materials from the packaging to foods is the focus of the present article. A major experimental program using eight polymer-migrant systems is described. Migration was measured to food-simulating liquids (FSL) and to foods. Accelerated tests were conducted with FSL under FDA guidelines conditions so as to develop correlations between such data and those found using foods under normal storage temperatures and shelf lives. In the majority of tests, the migration was found to be approximately proportional to the square root of time, to increase significantly with a rise in temperature, and to be proportional to the initial concentration of migrant in the polymer. Stirring in the FSL or food phase was generally not important except for the system involving dioctyl adipate migrating from polyvinyl chloride film. In some instances, after a period of time, migration rates became very low, and this effect was attributed to saturating the FSL or food phase with migrant. The foods comprised a variety of types, including liquid, semisolid, solid, and dry; both oily and aqueous foods were included. The physical steps involved in migration include the diffusion of the migrant from the interior of the film to the surface, where it can dissolve in the external FSL or food phase. The nature of the FSL or food is shown to be very important in that components can penetrate the polymer and dramatically increase migration rates. Consistent with the FDA guidelines in effect at the time of this study, testing was performed with five FSL (water, 3% acetic acid, 8% and 50% ethanol, and n-heptane) at 49 degrees C. Detailed comparisons were made between the migrations to foods and to FSL; following are the more relevant conclusions. (1) Three percent acetic acid showed no advantage over water as a food simulant even in those cases where the food could be considered acidic in nature. (2) Water, when used as an FSL at 49 degrees C for 5 days, overestimated migration in aqueous foods in about 75% of the cases. In some instances, however, the water phase became saturated with migrant. In other situations, this test protocol underpredicted migration--especially in those cases where there were components in the food that were able to penetrate into the polymer and enhance migration (such as orange juice).(ABSTRACT TRUNCATED AT 400 WORDS)

Plasticizer migration from polyvinyl chloride film to solvents and foods.

Polyvinyl chloride (PVC) films used for food wraps contain significant concentrations of plasticizers, along with other additives. The rate of migration of these plasticizers to foods and food-simulating solvents is the principal concern of this paper, which reviews prior experimental studies and presents new data for radiolabelled dioctyl adipate. Analytical models are described to correlate many of the data, criteria are presented for identifying the controlling step in the mechanism of transfer of plasticizer from PVC films into foods and food-simulating solvents, and tentative recommendations are offered for the selection of food simulants and for the type of experiment necessary to allow an unambiguous interpretation of the data.