Amino acid restriction modulates tumor growth, although effects on metastasis are poorly documented. We demonstrate that low levels of tyrosine (Tyr) and phenylalanine (Phe) suppress metastasis of B16-BL6 melanoma and that these effects are specific to these two amino acids. Weight loss and sustained low body weight in mice fed low Tyr and Phe diet do not contribute to the antimetastatic effects. Furthermore, methionine (Met) restriction, which decreased survival of mice inoculated i.p. with B16 melanoma, only slightly inhibited spontaneous metastasis compared to the dramatic inhibition during Tyr and Phe restriction. Tyr and Phe restriction inhibited spontaneous metastasis by impairing the ability of tumor cells to establish metastatic foci and not via differential tumor cell removal from the blood. Spontaneous metastasis is blocked by Tyr and Phe intervention even in mice with established lymph node tumors. Tumors isolated from mice fed low Tyr and Phe diet reinoculated into mice fed normal diet exhibited lower experimental metastatic potential, reflected by decreased formation of lung tumor colonies and increased survival of inoculated mice. This decrease in metastatic potential is not associated with tumor chemosensitivity. These findings indicate that Tyr and Phe restriction could become an important adjuvant to effective melanoma treatment.

Female C57BL/6 mice were fed a defined, pelleted diet and given 10% w/v or 20% w/v ethanol in their drinking water. Natural killer (NK) cell cytolytic activity was compared between water-drinking and ethanol-consuming mice and in mice that were also treated with polyinosinic-polycytidylic acid (poly I:C) to augment NK cell activity or with anti-NK1.1 antibody to decrease activity. NK cell cytolytic activity was not altered in mice given 10% ethanol, but was decreased in mice given 20% ethanol compared to water-drinking mice. Poly I:C treatment increased and anti-NK1.1 antibody treatment decreased NK cell activity in both water-drinking and 20% ethanol-consuming mice. Experimental and spontaneous metastases of B16-BL6 melanoma were evaluated as a function of the duration of ethanol consumption before tumor inoculation and as a function of altered NK cell activity. Experimental metastasis was inhibited after 4 and also after 6.5 weeks of ethanol exposure. Poly I:C treatment inhibited tumor lung colonization irrespective of ethanol consumption. Anti-NK1.1 antibody treatment increased metastasis, although to a lesser degree in mice consuming 10% ethanol. Spontaneous metastasis was inhibited in mice consuming 10% ethanol for 4 weeks, and in mice consuming 20% ethanol for 1 and 4 weeks before melanoma inoculation.

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

The northern section of the Upper Mississippi River supports a diverse macrobenthic assemblage. Distribution of this benthic fauna, benthic community structure, and factors which influence both of these phenomena in these upper pools are reviewed. Dumping of heavy loads of municipal and industrial wastes from the Minneapolis-St. Paul metropolitan area has severely stressed the benthic community. Once abundant, pollution-sensitive mayflies, Hexagenia bilineata and H. limbata, are noticeably absent, replaced by pollution-tolerant oligochaetes and midges (notably Chironomus). Harmful effects of this pollution are not restricted to the area immediately downstream from the Twin Cities. In Lake Pepin, the Hexagenia population has suffered a drastic decline. The benthic community is characterized by low species diversity and a dominant, pollution-tolerant Chironomusplumosus -Oligochaeta -Sphaeriidae -Hirudinea community complex. Farther south, effects of the high organic load which originates approximately 226 km upstream are ameliorated. Inundation of large, diverse land areas contributes to the great ecological diversity in Pools No. '7 and NO. 8. In Navigation Pool No. 7, benthic standing crops in the backwater pool areas (biomass range: 2.08-26.96 g mm2) exceed those in the main channel (biomass range: 0.05 -1.02 g rne2). Greater numbers of burrowing mayflies and mollusks were found in the pool areas. Of 131 taxa collected from 1976-1977 in Lake Onalaska, which occupies most of Pool No. 7, eight dominant groups -Oligochaeta, Hirudinea, Isopoda, Amphipoda, Lepidoptera, Diptera, Gastropoda, and Pelecypoda -accounted for 90-93% of the macroinvertebrates. In Pool No. 8, over half of the 144 benthic taxa collected during the summer of 1975 were insect nymphs and larvae. Oligochaetes were by far the most ubiquitous and dominant macroinvertebrates. Habitat preferences of particular benthic forms reflected distributional relationships between macroinvertebrates and physical-chemical conditions. Benthic production, in terms of total wet weight mm2 and macroinvertebrate density in each study area, was generally greater in the more eutrophic areas. However, fewer taxa were supported in these areas. These taxa were generally pollution-tolerant organisms, such as oligochaetes and certain chironomids, which were capable of burrowing into depositional-type substrates. More taxa and greater numbers of gill breathers and filter feeders, such as caddisflies, mayflies, stoneflies, and dipterans, were collected from less eutrophic areas.

Cancer chemotherapy frequently fails, because tumors develop multiple drug resistance (MDR). Pharmacological efforts to reverse this MDR phenotype and sensitize resistant tumor cells have utilized verapamil (VER) to inhibit p-glycoprotein function and buthionine sulfoximine (BSO) to inhibit glutathione synthesis. Our previous results indicate that restriction of two amino acids, tyrosine (Tyr) and phenylalanine (Phe), may potentially suppress the MDR phenotype. These results show that in vivo Tyr and Phe restriction improves the therapeutic response of a metastatic variant of B16-BL6 (BL6) murine melanoma to adriamycin (ADR) and B16 melanoma to levodopa methyl ester. We examine whether in vitro limitation of Tyr and Phe suppresses ADR resistance of BL6 cells and whether Tyr-Phe modulation of the MDR phenotype is applicable to other tumor types, particularly P388 murine leukemia. Mechanisms underlying Tyr-Phe modulation of ADR resistance are examined in the presence of VER and BSO, singly and in combination. Our results indicate that in vitro Tyr and Phe restriction has no effect on BL6 resistance to ADR. However, Tyr and Phe restriction does increase the sensitivity of ADR-resistant P388 cells to ADR without affecting drug efflux, ADR uptake, or glutathione levels. In addition, this enhanced ADR sensitivity of P388 cells is even more pronounced in the presence of BSO. Suppression of ADR resistance in P388-resistant cells by Tyr and Phe restriction indicates that Tyr- and Phe-mediated modulation of the MDR phenotype is possible and that Tyr and Phe restriction may be useful as a potential adjuvant to effective cancer chemotherapy.